Comparative Transcriptomic and Lipidomic Analyses of Human Male and Female Meibomian Glands Reveal Common Signature Genes of Meibogenesis.

Meibum is a lipid secretion that is produced by holocrine Meibomian glands (MGs). MGs are a specialized type of sebaceous glands that are embedded in the human eyelids. Chemically, meibum and sebum are different. A detailed characterization of lipidome and transcriptome of MG is required to deconvolute a complex and poorly characterized array of biosynthetic reactions (termed meibogenesis) that lead to formation of meibum. Changes in the composition and quality of meibum have been linked to various ocular disorders, some of which are more prevalent in males, while others in females. To establish the role of gender in meibogenesis in humans, we characterized MG transcriptomes and lipidomes of females and males, and identified signature genes of meibogenesis in both genders. Specimens of MG tissues were subjected to mRNA microarray analyses. Chemical composition of meibum samples was assessed chromatographically and mass spectrometrically. Both targeted and untargeted approaches were used. About 290 signature genes of meibogenesis were identified. The analyses of their expression patterns demonstrated no major differences between the genders. Lipid profiling of major classes of meibomian lipids, such as wax esters, cholesteryl esters, free cholesterol, (O)-acylated omega-hydroxy fatty acids (OAHFA), cholesteryl esters of OAHFA, and triacylglycerols, also demonstrated only minor (and random) differences in these lipids. The results of transcriptomic analyses correlated well with lipidomic data. Taken together, our data imply that in males and females, meibogenesis proceeds in a similar fashion, yielding secretions with similar, highly conserved, compositions. This finding is important for designing novel, gender-independent diagnostic and therapeutic approaches to various MG-related diseases and pathological conditions.

Effects of sex (or lack thereof) on meibogenesis in mice (Mus musculus): Comparative evaluation of lipidomes and transcriptomes of male and female tarsal plates.

The possible role of sex in the biosynthesis of lipids in the Meibomian glands (termed meibogenesis) remains unclear. To determine if there were any major sex-specific differences in the lipid composition of meibomian gland secretions (meibum) and gene expression patterns (GEP) related to meibogenesis, we conducted a study using healthy, age and diet-matched young adult wild-type C57BL/6J mice (2-2.5 month old). Tarsal plates (TP) were surgically excised from the eyelids of mice and subjected to transcriptomic and lipidomic analyses. The GEP were studied using mRNA microarrays. Lipids were extracted with organic solvents and analyzed using liquid chromatography and mass spectrometry. GEP in the TP of female and male mice demonstrated no statistically significant differences in the expression levels of the main protein-coding genes related to lipid metabolism and storage in general, and meibogenesis specifically (such as Elovl, Scd, Fads, Soat, Far, Awat, Acat, Lss, Dhcr, Hmgcr, Hmgcs, Dgat, Bckdh, Dbt, Fasn, and Plin, among others). The meibomian lipid profiles of female and male mice were virtually indistinguishable: all major lipids such as waxes, cholesteryl esters, cholesterol, (O)-acylated omega-hydroxy fatty acids (OAHFA), cholesteryl esters of OAHFA etc., were present in similar ratios. It seems that the major biosynthetic pathways in the Meibomian glands of male and female mice function in a similar fashion and produce secretions of the same overall chemical composition.

Dissecting lipid metabolism in meibomian glands of humans and mice: An integrative study reveals a network of metabolic reactions not duplicated in other tissues.

Lipids comprise the bulk of the meibomian gland secretion (meibum) which is produced by meibocytes. Complex arrays of lipogenic reactions in meibomian glands, which we collectively call meibogenesis, have not been explored on a molecular level yet. Our goals were to elucidate the possible biosynthetic pathways that underlie the generation of meibum, reveal similarities in, and differences between, lipid metabolism in meibomian glands and other organs and tissues, and integrate meibomian gland studies into the field of general metabolomics. Specifically, we have conducted detailed analyses of human and mouse specimens using genomic, immunohistochemical, and lipidomic approaches. Among equally highly expressed genes found in meibomian glands of both species were those related to fatty acid elongation, branching, desaturation, esterification, reduction of fatty acids to alcohols, and cholesterol biosynthesis. Importantly, corresponding lipid products were detected in meibum of both species using lipidomic approaches. For the first time, a cohesive, unifying biosynthetic scheme that connects genomic, lipidomic, and immunohistochemical observations is outlined and discussed.

Time-dependent degenerative transformations in the lipidome of chalazia.

The aim of this prospective study was to conduct histopathologic and lipidomic analyses of chalazia, in order to evaluate time-dependent changes in the lesion. Samples of surgically excised chalazia were collected over a period of 12 months from 10 patients (mean age 41 years; range, 23-58) with clinically diagnosed chalazia, who underwent scheduled surgery. The ages of chalazia varied from 2 to 28 weeks. To confirm the clinical diagnoses, the morphology of collected tissue samples was evaluated histologically after hematoxylin and eosin staining. The lipids from individual chalazia were analyzed by high-performance liquid chromatography-mass spectrometry and compared with authentic lipid standards and with the lipids of meibum collected from normal controls. We observed gradual, lesion age-dependent transformation of the lipidome of chalazia from an almost normal meibum-like composition to a very different kind of lipidome. A rapid initial increase in the free cholesterol content was followed by a gradual replacement of extremely long chain meibomian-type lipids with a mixture of shorter-chain cholesteryl esters of the C14-C18 family, triacylglycerols, ceramides, phospholipids and sphingomyelins. In addition, a rapid disappearance of wax esters and cholesteryl esters of (1-O)-acyl-omega-hydroxy fatty acids from the lipidome of aging chalazia was observed. Our results are indicative of dramatic, time-dependent changes in the lesion that may involve cholesterol as a trigger and/or a marker of subsequent degeneration of the meibomian lipidome. We hypothesize that early inhibition of these transformations may be useful in reversing the course of the disease.

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 systane and optive on aqueous tear evaporation in patients with dry eye disease.

OBJECTIVE: To compare the effect on aqueous tear (AT) evaporation rate of Systane and Optive at 30 min postinstillation in patients with dry eye. METHODS: In a crossover study of 20 patients with keratoconjunctivitis sicca, the evaporation rate of AT was measured. Evaporometry was used at two relative humidity (RH) ranges of 25% to 35% and 35% to 45%. The measurements were made at baseline (before the instillation of the study agent) and at 30 min after the instillation of 40 µL of either Systane or Optive per randomization assignment per visit with a 1-week interval between visits. RESULTS: No significant effects on AT evaporation rates at both RHs were found between study agents. CONCLUSIONS: In our study, neither Systane nor Optive has a significant impact on AT evaporation at 30 min postinstillation in patients with dry eye.

Meibomian lipid films and the impact of temperature.

PURPOSE: There is evidence that, in cold conditions, the temperature of human eyelids and of the ocular surface drops well below normal physiological levels. This may have a detrimental impact on the stability and functionality of the human tear film and the tear film lipid layer. The goal of this project was to quantitatively examine the possible impact of temperature on the latter. METHODS: Meibum samples were collected by using a soft-squeezing technique and were studied in a Langmuir trough. The obtained surface pressure and area isotherms were analyzed to determine the biophysical parameters of thin meibomian lipid film (MLF): the lift-off area, collapse pressure, two-dimensional elasticity, and hysteresis and their dependence on temperature. RESULTS: MLF was found to be highly susceptible to changes in temperature. At temperatures below the physiological level, the MLF became stiff and shrank considerably. The shrinkage left a large portion of the air-water interface uncovered with lipid molecules. The effect was shown to be reversible. On reheating, the lipids melted and respread to restore the original film. There was a fundamental difference observed between three-dimensional melting of dry meibum in bulk and the two-dimensional melting in MLF at the air-water interface. Bulk meibum melted in a narrower temperature range and showed a much higher cooperativity of melting. CONCLUSIONS: Temperature critically influences MLF. Low temperature leads to stiffening of the film, which loses its ability to form continuous layers at the air-water interface. These effects were shown be of a cooperative nature, manifesting in relatively narrow concentration and temperature ranges.

Corneal thickness association with ocular and corneal high-order aberrations.

PURPOSE: To investigate the association among central corneal thickness, peripheral corneal thickness (PCT), and wavefront aberrations in the anterior cornea, posterior cornea and the whole eye in myopia. METHODS: Sixty-four eyes of 64 myopic subjects were evaluated with a Pentacam rotating Scheimpflug camera (Oculus, Inc., WA) corneal topographer for: (1) wavefront aberrations from the anterior and posterior corneal surface, (2) corneal thickness (central and peripheral), and (3) with a wavefront aberration-supported cornea ablation wavefront analyzer (Wavescan, Visx, Inc., Santa Clara, CA) for wavefront aberrations generated in the whole eye. Relationships between the wavefront aberrations and corneal thickness were analyzed. RESULTS: The mean age of subjects was 34.75 +/- 10.08 years. The central corneal thickness was 550.5 +/- 28.459 microm. The mean peripheral thickness was 629.9 +/- 32.1 microm. Central and PCTs were not significantly correlated with corneal or ocular high-order aberrations. Intraocular pressure was significantly correlated with ocular trefoil (r = -0.307, P=0.001). CONCLUSIONS: Central and PCT were not significantly associated with either anterior or posterior corneal Zernike aberrations; in addition, no association with the whole ocular wavefront aberrations was found.

Historical brief on composition of human meibum lipids.

Meibomian glands (MG) secrete an oily substance, meibum, that spreads across the ocular surface and mixes with secretions produced by other ocular structures to create a thin film. The protective efficacy of the tear film is believed to be related to the chemical composition of the lipid layer. We reviewed the literature describing the composition of human MG secretions and have provided an overview on methods of collecting meibum samples, methods of lipid analyses, and the results obtained in previous studies. The usefulness and quality of the data obtained about meibum depend on proper sampling and the analytical techniques used. Historically, several methods have been developed, which have yielded contradictory data regarding meibum sample collection and analytical techniques. Based on review of the literature, the major lipids present in meibum are of nonpolar origin: waxes, sterols, and sterol esters, followed by triacylglycerides and fatty acids. The amphiphilic lipids, diacylglycerides, were reported in fewer studies, and monoacylglycerides were reported in only two. Information on the composition of the polar lipids is more controversial. Meibum phospholipids were found in small amounts (16% or less) in some studies, but not in others. Thus, meibum is a complex mixture of lipid molecules. Historical analytical inconsistencies may be partly explained by limitations of past analytical procedures and by the consideration that the tear film lipid layer may have contributing sources other than meibum.

Human tear film and meibum. Very long chain wax esters and (O-acyl)-omega-hydroxy fatty acids of meibum.

Human meibum was targetly analyzed for the presence of intact wax esters (WEs) and related compounds by means of reverse-phase HPLC in combination with ion trap mass spectrometry. The major detected WEs were based on C(18:n) (n = 1-4) unsaturated FAs ranking in the following order of abundance: C(18:1)>C(18:2)>C(18:3)>C(18:4). The major fatty alcohols (FAls) found in WE were of saturated nature and varied from C(18:0) to C(28:0). The three most abundant species were C(18:1)-FA esters of C(24:0), C(25:0), and C(26:0)-FAl. Typically, a major compound based on C(18:1)-FA and a saturated FAl was accompanied by a few related compounds based on a C(18:2), C(18:3), and C(18:4)-FA. Contrary to previous reports, no epoxy-WEs or epoxy-FAs were detected in fresh and 1-year-old meibum samples. More than 20 (O-acyl)-omega-hydroxy-FAs (OAHFAs) were observed. The main detected OAHFAs were based on very long-chain omega-hydroxy-FA (C(30:1), C(32:1), and C(34:1)) acylated through their omega-hydroxyls by a C(18:1)-FA. Due to their amphiphilic anionogenic nature, OAHFAs may be responsible for stabilization of the tear film lipid layer by creating an interface between the vast pool of strictly nonpolar lipids of meibum (WEs, cholesteryl esters, etc.) and the aqueous subphase beneath it, a role previously attributed to phospholipids.

Assessment and impact of the time of day on aqueous tear evaporation in normal subjects.

OBJECTIVES: To determine the impact of the time of day on aqueous tear (AT) evaporation in normal subjects on two consecutive days. METHODS: In a controlled laboratory setting, morning and afternoon AT evaporation was tested in 19 normal subjects, at the same time of day on two consecutive days. Evaporometry was used at two ranges of relative humidity (RH) 25% to 35% and 35% to 45%. RESULTS: Mean AT evaporation rates were 0.069 +/- 0.024 for 25% to 35% RH and 0.049 +/- 0.018 for 35% to 45% (P = 0.001). There were significant differences for both RH between time of day (P < 0.05) on day 1, but not observed on day 2. Variation between days showed no difference for RH during the afternoon, but there was a difference during the morning (P = 0.042). CONCLUSIONS: The data are remarkably consistent between study days, but there is more fluctuation during the morning than in the afternoon. Therefore, to further standardize AT evaporation study protocol, we recommend perform evaporometry measurements during the afternoon rather than the morning, because our results showed less variability during the afternoon test between days.