Human Meibomian Gland Research Articles

Influence of Omega 3 and 6 Fatty Acids on Human Meibomian Gland Epithelial Cells.

Oral supplementation with omega 3 (ω-3) and/or 6 (ω-6) fatty acids (FAs) has been reported to alleviate the signs and symptoms of dry eye disease and to improve the expressibility and quality of meibum, in patients with meibomian gland dysfunction. We tested our hypothesis that these FA effects may reflect a direct FA action on human meibomian gland epithelial cells. Immortalized human meibomian gland epithelial cells (IHMGECs) were cultured with ω-3, ω-6, or both FAs together for up to 7 days in the presence or absence of serum. After FA exposure, cells were analyzed for lipid expression, lysosome content, and proliferative ability. Our research shows that ω-3 and ω-6 stimulate the accumulation of small neutral lipid-containing vesicles, but not lysosomes, in IHMGECs. This vesicular effect was associated with a 2.4- to 3.7-fold increase in the cellular content of triglycerides after ω-3 and ω-6 treatment, respectively. The combination of both FAs together also enhanced triglyceride levels. Of interest, culture of IHMGECs with ω-3 and azithromycin, a known inducer of IHMGEC differentiation, led to a significantly greater amount of total neutral lipids, relative to that found with azithromycin alone. Cellular exposure to the FAs did not alter the expression of free or esterified cholesterol, or phospholipids. Further, these FAs, alone or together, prevented the proliferation of IHMGECs in serum-free, but not serum-containing, media. Our findings support our hypothesis and demonstrate that ω-3 and ω-6 can act directly on IHMGECs to influence the quality and quantity of intracellular lipids.

Notch Signaling in Meibomian Gland Epithelial Cell Differentiation.

PurposeNotch1 was previously shown to play a critical role in murine meibomian gland function and maintenance. In this study, we have examined the expression and activation of Notch pathway in human meibomian gland epithelial cells in vitro.MethodsAn immortalized human meibomian gland epithelial cell (HMGEC) line was cultured under proliferative and differentiative conditions. Expression of Notch receptors and ligands were evaluated by quantitative PCR and Western blot. The effect of Notch inhibition and induction on oil production was also assessed.ResultsHuman meibomian gland epithelial cell expressed Notch1, Notch2, Notch3, Jagged1, Jagged2, Delta-like 1, and Delta-like 3. The level of cleaved (activated) Notch1 strongly increased with differentiation. The expression of Notch3 was inversely correlated with proliferation. Induction and inhibition of Notch1 led to an increase and decrease in the amount of oil production, respectively.ConclusionsNotch signaling appears to play an important role in human meibomian gland epithelial differentiation and oil production. This may provide a potential therapeutic pathway for treating meibomian gland dysfunction.

In Vivo Laser Scanning Confocal Microscopy of Human Meibomian Glands in Aging and Ocular Surface Diseases.

Meibomian glands (MGs) play a crucial role in the ocular surface homeostasis by providing lipids to the superficial tear film. Their dysfunction destabilizes the tear film leading to a progressive loss of the ocular surface equilibrium and increasing the risk for dry eye. In fact, nowadays, the meibomian gland dysfunction is one of the leading causes of dry eye. Over the past decades, MGs have been mainly studied by using meibography, which, however, cannot image the glandular structure at a cellular level. The diffusion of the in vivo laser scanning confocal microscopy (LSCM) provided a new approach for the structural assessment of MGs permitting a major step in the noninvasive evaluation of these structures. LSCM is capable of showing MGs modifications during aging and in the most diffuse ocular surface diseases such as dry eye, allergy, and autoimmune conditions and in the drug-induced ocular surface disease. On the other hand, LSCM may help clinicians in monitoring the tissue response to therapy. In this review, we summarized the current knowledge about the role of in vivo LSCM in the assessment of MGs during aging and in the most diffuse ocular surface diseases.

Effects of Insulin and High Glucose on Human Meibomian Gland Epithelial Cells.

Type 2 diabetes is a risk factor for meibomian gland dysfunction (MGD). We hypothesize that this diabetic impact is due, at least in part, to the effects of insulin resistance/deficiency and hyperglycemia on human meibomian gland epithelial cells (HMGECs). To begin to test this hypothesis, we examined whether insulin and high glucose influence immortalized (I) HMGECs. Immortalized HMGECs were cultured in serum-containing or -free media and treated with insulin, insulin-like growth factor-1 (IGF-1), IGF-1 receptor (R) blocking antibody, and glucose or mannitol for varying time periods. Specific proteins were detected by Western blots, cell proliferation was evaluated by manual cell counting and lipids were assessed with LipidTOX and high performance thin layer chromatography. We found that insulin induces a dose-dependent increase in phosphatidylinositide 3-kinase/Akt (AKT) signaling in IHMGECs. This effect involves the IGF-1R, but not the insulin receptor (IR), and is associated with a stimulation of cell proliferation and neutral lipid accumulation. In contrast, high glucose exposure alters cell morphology, causes a progressive cell loss, and significantly reduces the levels of IGF-1R, phospho (p)-AKT, Foxhead box protein O1 (FOXO1), and sterol-regulatory element binding protein (SREBP-1) in IHMGECs. Our data show that insulin stimulates, and that high glucose is toxic for, IHMGECs. These results support our hypothesis that insulin resistance/deficiency and hyperglycemia are deleterious for HMGECs and may help explain why type II diabetes is a risk factor for MGD.

In vitro effects of docosahexaenoic and eicosapentaenoic acid on human meibomian gland epithelial cells

To investigate the effect of ω-3 fatty acids on human meibomian gland epithelial cells (HMGECs, cell line) in vitro. HMGECs were stimulated with docosahexaenoic acid (DHA) or combinations with eicosapentaenoic acid (EPA) and acetyl sialic acid (ASA). Sudan III fat staining, viability and proliferation assays, electric cell-substrate impedance sensing, real-time PCR for gene expression of cyclooxygenase-2 and 15-lipoxygenase and ELISAs for resolvin D1 (RvD1), IFNγ, TNFα and IL-6 were applied. Lipid droplet accumulation and viability was increased by 100 μM DHA in the presence or absence of EPA in serum cultured HMGECs. In contrast, HMGECs cultured with DHA and EPA under serum-free conditions showed minimal lipid accumulation, decreased proliferation and viability. Normalized impedance was significantly reduced in serum-free cultured HMGECs when stimulated with DHA and EPA. HMGECs cultured in serum containing medium showed increased normalized impedance under DHA and EPA stimulation compared to DHA or EPA alone or controls. IL-6 and IFNγ were downregulated in HMGECs treated for 72 h with DHA and EPA. In general, TNFα, IFNγ and IL-6 levels were decreased after 72 h compared to 24 h in serum containing medium with or without DHA or EPA. The concentration of RvD1 was elevated 2-fold after DHA treatment. Cyclooxygenase-2 gene expression decreased compared to controls during DHA stimulation after 72 h. Treatment with DHA and ASA revealed a decreased 15-lipoxygenase gene expression which was reduced after three days of DHA incubation. DHA and EPA supplementation affected HMGECs in vitro and supported anti-inflammatory effects by influencing cytokine levels, decreasing COX-2 expression and increasing the production of RvD1.

Characterization of Wax Esters by Electrospray Ionization Tandem Mass Spectrometry: Double Bond Effect and Unusual Product Ions.

A series of different types of wax esters (represented by RCOOR') were systematically studied by using electrospray ionization (ESI) collision-induced dissociation tandem mass spectrometry (MS/MS) along with pseudo MS(3) (in-source dissociation combined with MS/MS) on a quadrupole time-of-flight (Q-TOF) mass spectrometer. The tandem mass spectra patterns resulting from dissociation of ammonium/proton adducts of these wax esters were influenced by the wax ester type and the collision energy applied. The product ions [RCOOH2](+), [RCO](+) and [RCO-H2O](+) that have been reported previously were detected; however, different primary product ions were demonstrated for the three wax ester types including: (1) [RCOOH2](+) for saturated wax esters, (2) [RCOOH2](+), [RCO](+) and [RCO-H2O](+) for unsaturated wax esters containing only one double bond in the fatty acid moiety or with one additional double bond in the fatty alcohol moiety, and (3) [RCOOH2](+) and [RCO](+) for unsaturated wax esters containing a double bond in the fatty alcohol moiety alone. Other fragments included [R'](+) and several series of product ions for all types of wax esters. Interestingly, unusual product ions were detected, such as neutral molecule (including water, methanol and ammonia) adducts of [RCOOH2](+) ions for all types of wax esters and [R'-2H](+) ions for unsaturated fatty acyl-containing wax esters. The patterns of tandem mass spectra for different types of wax esters will inform future identification and quantification approaches of wax esters in biological samples as supported by a preliminary study of quantification of isomeric wax esters in human meibomian gland secretions.

Serum-induced keratinization processes in an immortalized human meibomian gland epithelial cell line.

PurposeThe aim of this study was to evaluate a human meibomian gland epithelial cell line (HMGEC) as a model for meibomian gland (patho)physiology in vitro.MethodsHMGEC were cultured in the absence or presence of serum. Sudan III lipid staining, ultrastructural analysis and lipidomic analyses were performed. Impedance sensing, desmoplakin 1/2 mRNA and cytokeratin (CK) 1, 5, 6, 14 levels were evaluated. Serum containing medium supplemented with higher serum, glucose, an omega-3 lipid cocktail, eicosapentaenoic acid or sebomed medium were investigated for lipid accumulation and ultrastructural morphology.ResultsLipid droplet accumulation in HMGEC was induced by serum containing media after 1 day, but decreased over time. Cultivation in serum induced desmosome and cytokeratin filament formation. Desmoplakin 1/2 gene levels were significantly upregulated after 1d of serum treatment. Furthermore, the normalized impedance increased significantly. Lipidome analysis revealed high levels of phospholipids (over 50%), but very low levels of wax ester and cholesteryl esters (under 1%). Stimulation with eicosapentaenoic acid increased lipid accumulation after one day.ConclusionSerum treatment of HMGEC caused lipid droplet formation to some extent but also induced keratinization. The cells did not produce typical meibum lipids under these growth conditions. HMGEC are well suited to study (hyper)keratinization processes of meibomian gland epithelial cells in vitro.

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.

Azithromycin and MGD: Is it possible to turn an off‐label treatment into an on‐label therapy?

AbstractMeibomian gland dysfunction (MGD) is the primary cause of dry eye disease (DED), which afflicts hundreds of millions of people, predominantly women, and is a leading cause of patient visits to eye care practitioners. The impact of moderate to severe DED is comparable to conditions such as dialysis and severe angina, and is associated with significant pain, role limitations, low vitality and poor general health. There is no cure for MGD. The most frequent pharmaceutical treatment in the United States for the management of MGD is the off‐label use of topical azithromycin. This antibiotic is presumed to be effective because of its anti‐inflammatory and anti‐bacterial actions, which may suppress the MGD‐associated posterior blepharitis and growth of lid bacteria. We hypothesize that azithromycin can act directly on human meibomian gland epithelial cells to stimulate their differentiation, enhance the quality and quantity of their lipid production, promote their holocrine secretion and ameliorate MGD pathophysiology. Our results support our hypotheses and may help lead to the approval of topical azithromycin as a safe and effective therapy for human MGD and thereby improve the quality of life of countless people throughout the world. Commercial interest

The combined effect of azithromycin and insulin-like growth factor-1 on cultured human meibomian gland epithelial cells.

Meibomian gland dysfunction (MGD) is the leading cause of dry eye disease, a prevalent disorder severely affecting patients' quality of life but has no cure. We have discovered that azithromycin, a topical antibiotic used off-label to treat MGD-associated posterior blepharitis, directly acts on the human meibomian gland epithelial cells (HMGECs) to promote their differentiation, and in doing so, reduces cell proliferation. We have also found that insulin-like growth factor-1 (IGF-1), a drug approved by the Food and Drug Administration primarily used to treat dwarfism, stimulates the proliferation and lipid accumulation in these cells. We hypothesize that the combination of azithromycin and IGF-1 will promote cellular differentiation and lipid accumulation, while preserving the normal proliferation of HMGECs. We cultured immortalized HMGECs with vehicle, 10 nM IGF-1, 10 μg/mL azithromycin, or a combination of IGF-1 and azithromycin for 5 to 13 days. Cells were evaluated for intracellular neutral lipids and lysosome accumulation by different staining methods; lipid composition of cell lysates were analyzed using high-performance thin-layer chromatography; proteins of interest (sterol regulatory element binding protein-1 [SREBP-1], cyclins B1 and D1) were measured by immunoblotting, and cell numbers were counted using a hemocytometer. Our findings demonstrate that the combination of azithromycin and IGF-1 promotes the differentiation and lipid accumulation of HMGECs, while preserving their normal proliferation rate. This combined treatment also increased the levels of neutral lipids, phospholipids, and SREBP-1, and restored cyclin B1 content to control amounts. Our results support our hypothesis, and this combination regime may represent a unique and effective treatment of MGD.

Serum-induced differentiation of human meibomian gland epithelial cells.

We hypothesize that culturing immortalized human meibomian gland epithelial cells in serum-containing medium will induce their differentiation. The purpose of this investigation was to begin to test our hypothesis, and explore the impact of serum on gene expression and lipid accumulation in human meibomian gland epithelial cells. Immortalized and primary human meibomian gland epithelial cells were cultured in the presence or absence of serum. Cells were evaluated for lysosome and lipid accumulation, polar and neutral lipid profiles, and gene expression. Our results support our hypothesis that serum stimulates the differentiation of human meibomian gland epithelial cells. This serum-induced effect is associated with a significant increase in the expression of genes linked to cell differentiation, epithelium development, the endoplasmic reticulum, Golgi apparatus, vesicles, and lysosomes, and a significant decrease in gene activity related to the cell cycle, mitochondria, ribosomes, and translation. These cellular responses are accompanied by an accumulation of lipids within lysosomes, as well as alterations in the fatty acid content of polar and nonpolar lipids. Of particular importance, our results show that the molecular and biochemical changes of immortalized human meibomian gland epithelial cells during differentiation are analogous to those of primary cells. Overall, our findings indicate that immortalized human meibomian gland epithelial cells may serve as an ideal preclinical model to identify factors that control cellular differentiation in the meibomian gland.

The Effects of Insulin-like Growth Factor 1 and Growth Hormone on Human Meibomian Gland Epithelial Cells

A phase 1 study has reported that dry eye disease is the most common adverse effect of human exposure to the antibody figitumumab, an anticancer drug that prevents insulin-like growth factor 1 (IGF-1) from binding to its receptor. We hypothesized that the mechanism underlying this effect is the inhibition of IGF-1 action in epithelial cells of the meibomian gland. To test the hypothesis that IGF-1 stimulates meibomian gland function in vitro and to examine whether growth hormone, a closely related hormone of IGF-1, has the same effect. DESIGN, SETTING, AND MATERIAL: Immortalized human meibomian gland epithelial cells were cultured in the presence or the absence of IGF-1, growth hormone, and an IGF-1 receptor-blocking antibody. Signaling pathways, cell proliferation, neutral lipid staining, and a key protein involved in lipid biogenesis were evaluated. Application of IGF-1 and growth hormone to human meibomian gland epithelial cells. Immunoblotting, cell counting, and neutral lipid staining. RESULTS Insulin-like growth factor 1 activated the phosphoinositol 3-kinase/Akt and forkhead box O1 pathways (showing a dose-dependent effect on immunoblotting), stimulated cellular proliferation (about 1.8-fold increase in cell number), increased sterol regulatory element-binding protein 1 expression (about 3-fold increase on immunoblotting), and promoted lipid accumulation in human meibomian gland epithelial cells (about 2-fold increase in lipid staining). These IGF-1 actions, which may be blocked by cotreatment with the anti-IGF-1 antibody, were accompanied by inconsistent effects on extracellular signal-regulated kinase phosphorylation. We were not able to demonstrate activation of Akt, forkhead box O1, extracellular signal-regulated kinase, Janus kinase 2, or signal transducers and activators of transcription 5, induced cell proliferation, or lipid accumulation in these cells by growth hormone application. Our results support the hypothesis that IGF-1 acts on human meibomian gland epithelial cells and may explain why treatment with figitumumab, the IGF-1 inhibitor, causes dry eye disease. Ophthalmic care for dry eye disease may be needed when patients with cancer undergo treatment with drugs that inhibit IGF-1 action.

What drives Meibomian gland disease?

The meibomian gland dysfunction (MGD) is the leading cause of dry eye disease (DED) throughout the world. The investigation of MGD lacks suitable in vivo and in vitro models. In 2010 a human meibomian gland epithelial cell line (HMGEC) was established, so far the only available meibomian gland cell line. The characterization of HMGEC is of major importance to clarify its suitability for studying the meibomian gland (patho)physiology in vitro. The current culture protocol and new concepts of HMGEC culture will be compared. Hormones are believed to be a key factor in meibomian gland dysfunction thus HMGEC responsiveness to hormone stimulation is crucial to elucidate the hormonal influence on the meibomian gland. This review will summarize current findings about HMGEC and discuss its role in the meibomian gland dysfunction research.

Three-dimensional volumetric human meibomian gland investigation using polarization-sensitive optical coherence tomography

In this study, polarization-sensitive optical coherence tomography (PS-OCT) capable of providing polarization contrasts such as phase retardation and degree of polarization uniformity (DOPU) was used for visualizing human meibomian glands (MGs) and investigating morphological characteristics of them. Especially, with the help of the DOPU contrast, MGs were exclusively extracted from the volumetric OCT image. In vivo PS-OCT measurements were performed on the upper eyelids of different age groups. From these measurements, different age-dependent aspects of the MG structure were also observed. Based on these observations, it can be inferred that the PS-OCT system has the potential for clinical diagnosis and investigation of MG-related dry eye diseases like MG dysfunction (MGD) and acinar atrophy.

One man's poison is another man's meat: Using azithromycin-induced phospholipidosis to promote ocular surface health

Drug-induced phospholipidosis (PLD) is a common adverse effect which has led to the termination of clinical trials for many candidate pharmaceuticals. However, this lipid-inducing effect may be beneficial in the treatment of meibomian gland dysfunction (MGD). MGD is the major cause of dry eye disease (DED), which affects 40 million people in the USA and has no cure. Azithromycin (AZM) is a PLD-inducing antibiotic that is used off-label to treat MGD, and is presumably effective because it suppresses the MGD-associated conjunctival inflammation (i.e. posterior blepharitis) and growth of lid bacteria. We hypothesize that AZM can act directly to promote the function of human meibomian gland epithelial cells by inducing PLD in these cells, characterized by the accumulation of lipids and lysosomes. Immortalized human meibomian gland epithelial cells (HMGEC) were cultured with or without azithromycin for 5 days. Cells were evaluated for cholesterol (Filipin) and neutral lipid (LipidTox) staining, as well as the appearance of lysosomes (LysoTracker) and lamellar bodies (transmission electron microscopy, TEM). The lipid composition of cellular lysates was analyzed by high performance thin-layer chromatography. Our findings demonstrate that AZM stimulates the accumulation of free cholesterol, neutral lipids and lysosomes in HMGEC. This AZM-induced increase of neutral lipid content occurred predominantly within lysosomes. Many of these vesicles appeared to be lamellar bodies by TEM, which is the characteristic of PLD. Our findings also show that AZM promotes an accumulation of free and esterified cholesterol, as well as phospholipids in HMGECimmortalized. Our results support our hypothesis and confirm the beneficial effect of PLD induced by AZM on HMGEC. Our discovery reveals a new potential use of PLD-inducing drugs, and makes this adverse effect a beneficial effect.

Effect of Azithromycin on Lipid Accumulation in Immortalized Human Meibomian Gland Epithelial Cells

Meibomian gland dysfunction (MGD) is believed to be the leading cause of dry eye disease (DED), which afflicts tens of millions Americans (1). Of particular interest, the most common pharmaceutical treatment for the management of MGD in the United Statesis the off-label use of topical azithromycin (2). This macrolide antibiotic is presumed to be effective because of its anti-inflammatory and anti-bacterial actions, which may suppress the MGD-associated posterior blepharitis and growth of lid bacteria (3). However, there are no published, peer-reviewed data demonstrating that azithromycin has the ability to act directly on the human meibomian gland to enhance this tissue’s function, and to ameliorate the pathophysiology of MGD. We hypothesize that azithromycin can act directly on human meibomian gland epithelial cells to stimulate their differentiation, enhance the quality and quantity of their lipid production, and promote their holocrine secretion. Our purpose was to begin to test our hypothesis.

The Influence of 13-cisRetinoic Acid on Human Meibomian Gland Epithelial Cells

Meibomian gland dysfunction (MGD) is a primary cause of dry eye disease. One of the risk factors for MGD is exposure to 13-cis retinoic acid (13-cis RA), a metabolite of vitamin A. However, the mechanism is not well understood. We hypothesize that 13-cis RA inhibits cell proliferation, promotes cell death, alters gene and protein expressions, and attenuates cell survival pathways in human meibomian gland epithelial cells. To test our hypotheses, immortalized human meibomian gland epithelial cells were cultured with or without 13-cis RA for varying doses and time. Cell proliferation, cell death, gene expression, and proteins involved in proliferation/survival and inflammation were evaluated. We found that 13-cis RA inhibited cell proliferation, induced cell death, and significantly altered the expression of 6726 genes, including those involved in cell proliferation, cell death, differentiation, keratinization, and inflammation, in human meibomian gland epithelial cells. Further, 13-cis RA also reduced the phosphorylation of Akt and increased the generation of interleukin-1β and matrix metallopeptidase 9. Exposure to 13-cis RA inhibits cell proliferation, increases cell death, alters gene expression, changes signaling pathways, and promotes inflammatory mediator and protease expression in meibomian gland epithelial cells. These effects may be responsible, at least in part, for the 13-cis RA-related induction of MGD.

In Vivo 3D Meibography of the Human Eyelid Using Real Time Imaging Fourier-Domain OCT

Recently, we reported obtaining tomograms of meibomian glands from healthy volunteers using commercial anterior segment optical coherence tomography (AS-OCT), which is widely employed in clinics for examination of the anterior segment. However, we could not create 3D images of the meibomian glands, because the commercial OCT does not have a 3D reconstruction function. In this study we report the creation of 3D images of the meibomian glands by reconstructing the tomograms of these glands using high speed Fourier-Domain OCT (FD-OCT) developed in our laboratory. This research was jointly undertaken at the Department of Ophthalmology, Seoul St. Mary's Hospital (Seoul, Korea) and the Advanced Photonics Research Institute of Gwangju Institute of Science and Technology (Gwangju, Korea) with two healthy volunteers and seven patients with meibomian gland dysfunction. A real time imaging FD-OCT system based on a high-speed wavelength swept laser was developed that had a spectral bandwidth of 100 nm at the 1310 nm center wavelength. The axial resolution was 5 µm and the lateral resolution was 13 µm in air. Using this device, the meibomian glands of nine subjects were examined. A series of tomograms from the upper eyelid measuring 5 mm (from left to right, B-scan) × 2 mm (from upper part to lower part, C-scan) were collected. Three-D images of the meibomian glands were then reconstructed using 3D “data visualization, analysis, and modeling software”. Established infrared meibography was also performed for comparison. The 3D images of healthy subjects clearly showed the meibomian glands, which looked similar to bunches of grapes. These results were consistent with previous infrared meibography results. The meibomian glands were parallel to each other, and the saccular acini were clearly visible. Here we report the successful production of 3D images of human meibomian glands by reconstructing tomograms of these glands with high speed FD-OCT.

Differential scanning calorimetric evaluation of human meibomian gland secretions and model lipid mixtures: Transition temperatures and cooperativity of melting

Meibomian gland secretions (or meibum) are produced by holocrine meibomian glands and are secreted in melted form onto the ocular surface of humans and animals to form a protective tear film lipid layer (TFLL). Its protective effect strongly depends on the composition and, hence, thermotropic behavior of meibum. The goal of our study was to quantitatively evaluate the melting characteristics of human meibum and model lipid mixtures using differential scanning microcalorimetry. Standard calorimetric parameters, e.g. changes in calorimetric enthalpy, transition temperatures Tm, cooperativity of melting, etc. were assessed. We found that thermotropic behavior of meibum resembled that of relatively simple mixtures of unsaturated wax esters, but showed a lower change in calorimetric enthalpy, which can be indicative of a looser packing of lipids in meibum compared with pure standards and their simple mixtures. The cooperativity of melting of meibomian lipids was comparable to that of an equimolar mixture of four oleic-acid based wax esters. We demonstrated that the phase transitions in meibum start at about 10–15°C and end at 35–36°C, with Tm being about 30°C. The highly asymmetrical shape of the thermotropic peak of meibum is important for the physiology and biophysics of TFLL.

Effect of Growth Factors on the Proliferation and Gene Expression of Human Meibomian Gland Epithelial Cells

We hypothesize that growth factors, including epidermal growth factor (EGF) and bovine pituitary extract (BPE), induce proliferation, but not differentiation (e.g., lipid accumulation), of human meibomian gland epithelial cells. We also hypothesize that these actions involve a significant upregulation of genes linked to cell cycle processes, and a significant downregulation of genes associated with differentiation. Our objective was to test these hypotheses. Immortalized human meibomian gland and conjunctival epithelial cells were cultured for varying time periods in the presence or absence of EGF, BPE, EGF + BPE, or serum, followed by cell counting, neutral lipid staining, or RNA isolation for molecular biological procedures. Our studies show that growth factors stimulate a significant, time-dependent proliferation of human meibomian gland epithelial cells. These effects are associated with a significant upregulation of genes linked to cell cycle, DNA replication, ribosomes, and translation, and a significant decrease in those related to cell differentiation, tissue development, lipid metabolic processes, and peroxisome proliferator-activated receptor signaling. Serum-induced differentiation, but not growth factor-related proliferation, elicits a pronounced lipid accumulation in human meibomian gland epithelial cells. This lipogenic response is unique, and is not duplicated by human conjunctival epithelial cells. Our results demonstrate that EGF and BPE stimulate human meibomian gland epithelial cells to proliferate. Further, our findings show that action is associated with an upregulation of cell cycle and translation ontologies, and a downregulation of genetic pathways linked to differentiation and lipid biosynthesis.