Physiological Effects of Soat1 Inactivation on Homeostasis of the Mouse Ocular Surface.

Purpose: Soat1/SOAT1 have been previously reported to be critical for the biosynthesis of cholesteryl esters (CEs) in the mouse Meibomian glands (MGs) as the loss of function led to an arrest of CE production and a substantial accumulation of nonesterified cholesterol in the meibum, causing an increase in its melting temperature. The purpose of this study was to further investigate the role of Soat1 in meibogenesis and ocular surface physiology. Methods: The mouse ocular features of knockout Soat1-/- and wild type (WT) mice were studied using various ophthalmic and histological techniques, mouse lipidomes were monitored using liquid chromatography/mass spectrometry, whereas their transcriptomes were compared to characterize the effects of the mutation on the gene expression profiles in the MG and cornea. Results: Soat1-/- mice displayed increased tear production and severe corneal abnormalities, such as corneal thinning, (neo)vascularization, ulceration, and opacification that progressed with aging. Transcriptomic analyses led to identification of a range of significantly disrupted pathways, which included general and specific lipid metabolism-related pathways, keratinization, angiogenesis/(neo)vascularization, muscle contraction, and several other pathways. In addition, histological and histochemical experiments revealed morphological changes in the MG, cornea, and conjunctiva in Soat1-/- mice. Notably, the mRNA microarray expression level of Soat1 in WT MGs (log2 17.5) was 1000 × of that in the mouse cornea (log2 7.5). Conclusions: These findings suggest a direct involvement of Soat1/SOAT1 in MGs in maintaining ocular surface homeostasis, in general, and corneal health, specifically.

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.

Dysregulation of Lipid Metabolism in Aging Meibomian Glands and Its Molecular Markers.

The main function of exocrine Meibomian glands (MGs) is to produce a lipid-rich secretion called meibum which plays a critical role in maintaining the ocular surface homeostasis of humans and most mammals. The chemical composition of meibum, and its quantity produced by MGs, largely determine whether it can fulfill its role successfully. Aging was frequently associated with the onset of various MG-related pathologies. The goal of this study was to determine how aging affects the chemical composition and quantity of meibum in mice, and identify possible molecular markers of aging. Unbiased, untargeted and targeted lipidomic evaluation of mouse MG lipids was conducted using liquid chromatography-high-resolution mass spectrometry, and the results were analyzed using Principal Component, Orthogonal Projections to Latent Structures Discriminant, and Partial Least Square Discriminant Analyses. We found that aging leads to dysregulation of lipid metabolism in MGs, changing the ratios of major classes of MG lipids (such as wax esters, triacylglycerols, and phospholipids) in a progressive manner. Several lipid species that belong to these groups of MG lipids are proposed as clear markers of aging in a mouse model.

Comparative Biophysical Study of Meibomian Lipids of Wild Type and Soat1-Null Mice: Implications to Meibomian Gland Dysfunction and Dry Eye Disease.

Purpose: The biophysical roles of Meibomian lipids (MLs) played in health and meibomian gland dysfunction (MGD) are still unclear. The purpose of this research is to establish the composition-structure-functional correlations of the ML film (MLF) using Soat1-null mice and comprehensive in vitro biophysical simulations. Methods: MLs were extracted from tarsal plates of wild type (WT) and Soat1 knockout (KO) mice. The chemical composition of ML samples was characterized using liquid chromatography - mass spectrometry. Comprehensive biophysical studies of the MLFs, including their dynamic surface activity, interfacial rheology, evaporation resistance, and ultrastructure and topography, were performed with a novel experimental methodology called the constrained drop surfactometry. Results: Soat1 inactivation caused multiple alternations in the ML profile. Compared to their WT siblings, the MLs of KO mice were completely devoid of cholesteryl esters (CEs) longer than C18 to C20, but contained 7 times more free cholesterol (Chl). Biophysical assays consistently suggested that the KO-MLF became stiffer than that of WT mice, revealed by reduced film compressibility, increased elastic modulus, and decreased loss tangent, thus causing more energy loss per blinking cycle of the MLF. Moreover, the KO mice showed thinning of their MLF, and reduced evaporation resistance. Conclusions: These findings delineated the composition-structure-functional correlations of the MLF and suggested a potential biophysical function of long-chain CEs in optimizing the surface activity, interfacial rheology, and evaporation resistance of the MLF. This study may provide novel implications to pathophysiological and translational understanding of MGD and dry eye disease.

Forward genetic screening using fundus spot scale identifies an essential role for Lipe in murine retinal homeostasis.

Microglia play a role in the pathogenesis of many retinal diseases. Fundus spots in mice often correlate with the accumulation of activated subretinal microglia. Here we use a semiquantitative fundus spot scoring scale in combination with an unbiased, state-of-the-science forward genetics pipeline to identify causative associations between chemically induced mutations and fundus spot phenotypes. Among several associations, we focus on a missense mutation in Lipe linked to an increase in yellow fundus spots in C57BL/6J mice. Lipe-/- mice generated using CRISPR-Cas9 technology are found to develop accumulation of subretinal microglia, a retinal degeneration with decreased visual function, and an abnormal retinal lipid profile. We establish an indispensable role of Lipe in retinal/RPE lipid homeostasis and retinal health. Further studies using this new model will be aimed at determining how lipid dysregulation results in the activation of subretinal microglia and whether these microglia also play a role in the subsequent retinal degeneration.

Sdr16c5 and Sdr16c6 control a dormant pathway at a bifurcation point between meibogenesis and sebogenesis.

Genes Sdr16c5 and Sdr16c6 encode proteins that belong to a superfamily of short-chain dehydrogenases/reductases (SDR16C5 and SDR16C6). Simultaneous inactivation of these genes in double-KO (DKO) mice was previously shown to result in a marked enlargement of the mouse Meibomian glands (MGs) and sebaceous glands, respectively. However, the exact roles of SDRs in physiology and biochemistry of MGs and sebaceous glands have not been established yet. Therefore, we characterized, for the first time, meibum and sebum of Sdr16c5/Sdr16c6-null (DKO) mice using high-resolution MS and LC. In this study, we demonstrated that the mutation upregulated the overall production of MG secretions (also known as meibogenesis) and noticeably altered their lipidomic profile, but had a more subtle effect on sebogenesis. The major changes in meibum of DKO mice included abnormal accumulation of shorter chain, sebaceous-type cholesteryl esters and wax esters (WEs), and a marked increase in the biosynthesis of monounsaturated and diunsaturated Meibomian-type WEs. Importantly, the MGs of DKO mice maintained their ability to produce typical extremely long chain Meibomian-type lipids at seemingly normal levels. These observations indicated preferential activation of a previously dormant biosynthetic pathway that produce shorter chain, and more unsaturated, sebaceous-type WEs in the MGs of DKO mice, without altering the elongation patterns of their extremely long chain Meibomian-type counterparts. We conclude that the Sdr16c5/Sdr16c6 pair may control a point of bifurcation in one of the meibogenesis subpathways at which biosynthesis of lipids can be redirected toward either abnormal sebaceous-type lipidome or normal Meibomian-type lipidome in WT mice.

Primary cilia control cellular patterning of Meibomian glands during morphogenesis but not lipid composition.

Meibomian glands (MGs) are modified sebaceous glands producing the tear film's lipids. Despite their critical role in maintaining clear vision, the mechanisms underlying MG morphogenesis in development and disease remain obscure. Cilia-mediate signals are critical for the development of skin adnexa, including sebaceous glands. Thus, we investigated the role of cilia in MG morphogenesis during development. Most cells were ciliated during early MG development, followed by cilia disassembly during differentiation. In mature glands, ciliated cells were primarily restricted to the basal layer of the proximal gland central duct. Cilia ablation in keratine14-expressing tissue disrupted the accumulation of proliferative cells at the distal tip but did not affect the overall rate of proliferation or apoptosis. Moreover, impaired cellular patterning during elongation resulted in hypertrophy of mature MGs with increased meibum volume without altering its lipid composition. Thus, cilia signaling networks provide a new platform to design therapeutic treatments for MG dysfunction.

Probing dietary triacylglycerol metabolism and meibogenesis in mice: A stable isotope-labeled tracer liquid chromatography-tandem mass spectrometry study.

Exocrine meibomian glands (MGs) play a central role in the ocular physiology and biochemistry by producing in situ and, mostly, de novo a secretion (meibum), which is composed of a complex mixture of homologous lipids of various classes, in a metabolic pathway termed meibogenesis. Recent in vivo experiments with a number of mouse models demonstrated that inactivation of any of the major genes of meibogenesis led to alterations in the lipid composition of meibum and severe ocular and MG abnormalities that replicated various human ocular pathologies. However, the role of dietary lipids in meibogenesis, and in the onset and/or alleviation of these diseases, remains controversial. To uncover the role of dietary lipids, the metabolic transformations of a dietary lipid tracer-stable isotope-labeled glyceryl tri(oleate-1,2,3,7,8-13C5) (13C15-TO)-were investigated using liquid chromatography-high-resolution mass spectrometry. We demonstrated that major metabolic transformations of the tracer occurred in the stomach and small intestines where 13C15-TO underwent immediate and extensive transesterification into 13C5- and 13C10-substituted triacylglycerols of various lengths, giving a mixture of 13C-labeled compounds that remain virtually unchanged in the mouse plasma, liver, and white adipose tissue but were almost undetectable in the feces. Importantly, the tracer and its metabolites were virtually undetectable in MGs, even after 4 weeks of daily supplementation. Notably, unbiased principal component analysis of the data revealed no measurable changes in the overall chemical composition of meibum after the treatment, which implies no direct effect of dietary triacylglycerols on meibogenesis, and left their systemic effects as the most likely mechanism.

Dynamic Changes in the Gene Expression Patterns and Lipid Profiles in the Developing and Maturing Meibomian Glands.

Meibomian glands (MGs) and their holocrine secretion-meibum-play crucial roles in the physiology of the eye, providing protection from environmental factors and desiccation, among other functions. Importantly, aging was implicated in the deterioration of the morphology and functions of MGs, and the quantity and quality of meibum they produce, leading to a loss of its protective properties, while the meibum of young individuals and experimental animals provide ample protection to the eye. Currently, the molecular mechanisms of meibum biosynthesis (termed meibogenesis) are not fully understood. To characterize the physiological changes in developing and maturing MGs, we studied the lipidomes and transcriptomes of mouse MGs ranging from newborns to adults. The results revealed a gradual increase in the critical genes of meibogenesis (such as Elovl3, Elovl4, Awat2, and Soat1, among others) that positively correlated with the biosynthesis of their respective lipid products. The MG transcriptomes of young and adult mice were also analyzed using single-cell RNA sequencing. These experiments revealed the existence of multiple unique populations of MG cells (meibocytes, epithelial cells, and others) with specific combinations of genes that encode meibogenesis-related proteins, and identified clusters and subclusters of cells that were tentatively classified as meibocytes at different stages of differentiation/maturation, or their progenitor cells. A hypothesis was formulated that these cells may produce different types of lipids, and contribute differentially to the Meibomian lipidome.

Novel Lipids of the Rabbit Harderian Gland Improve Tear Stability in an Animal Model of Dry Eye Disease.

Purpose: Instability of the tear film leads to evaporative dry eye disease (EDED), but the Harderian gland in some terrestrial vertebrates may produce novel lipids that stabilize the tear film and protect against dry eye. Here, the nonpolar lipids in the Harderian gland and tears of the rabbit but absent in human tears were identified and tested in preclinical studies to determine whether they could treat severe EDED. Methods: Lipids were identified primarily by atmospheric pressure chemical ionization mass spectrometry (MS) and fragmentation MS/MS. An identified lipid was synthesized and formulated as an emulsion and as a cyclodextrin (CD) clathrate. Following doses with test agents and controls, tear film breakup time (TBUT), tear production, corneal fluorescein staining, macrophage infiltration, and goblet cell survival were measured using standard tests at 0, 2 and 4 weeks in an animal model of EDED. Results: The lipid emulsion increased TBUT (P < 0.01) and tear production (P < 0.05), while it decreased corneal staining (P < 0.01) compared to controls. The lipid CD formulation increased TBUT (P < 0.05) and tear production (P < 0.05) but had no significant effect on the remaining test parameters. There were no differences in macrophage infiltration and conjunctival impression cytology scores between the formulations and their vehicle controls. Conclusions: Lipids in the rabbit Harderian gland and tears differ from those identified in human meibum and tears. These unique rabbit lipids may confer a protective effect against EDED and, as supplements to human tears, fulfill a similar role.

Effects of Aging on Human Meibum.

Purpose: The purpose of this study was to determine if aging affects meibum lipid composition in non-meibomian gland dysfunction (MGD)/non-dry eye (DE) population. Aging has been repeatedly linked to pathological changes in various tissues and organs, including the onset of MGD and DE, in a number of clinical and population-wide surveys. Both conditions have been associated with abnormal meibum secretion and composition, among other factors. However, the chemical basis for such a connection has not been established yet. Methods: To identify and characterize possible changes in the meibum and meibogenesis with aging, lipidomic analyses of meibum samples collected from human subjects of two age groups - young (29 ± 5 years, n = 21) and elderly (68 ± 7 years, n = 29) - with similar male to female ratios in each group were conducted. Intact lipid species from major lipid groups of meibum (such as wax esters, cholesteryl esters, free cholesterol, triacylglycerols, etc.) were compared using lipidome-wide untargeted (such as Principal Component Analysis) and targeted (such as Orthogonal Projections to Latent Structures Discriminant Analysis) approaches, along with focused analyses of specific lipid species in liquid-chromatography mass spectrometry (LC-MS) and tandem mass spectrometry (MS-MS) experiments. Results: Extremely high similarities of meibum lipids in the two age groups were observed, with only minor changes in the individual lipid species. The magnitude of the intergroup variability for tested lipid species was comparable to the intragroup variability for the same meibum components. No statistically significant differences in the lipid esterification, elongation, and unsaturation patterns were observed. Conclusions: Chronological aging itself seems to have only minor effect on meibogenesis in healthy, non-MGD/non-DE subjects.

Differential effects of dietary cholesterol and triglycerides on the lipid homeostasis in Meibomian glands.

Exocrine Meibomian glands (MG) play a central role in the ocular surface physiology by producing meibum - a lipid secretion composed of cholesteryl esters (CE), cholesterol (Chl), triacylgycerols (TAG), waxes and other types of lipids. MG were previously shown to synthesize Meibomian lipids (ML) in situ via a complex array of reactions termed meibogenesis. However, questions remain about the role of dietary lipids in meibogenesis. To establish if dietary Chl (DC) and TAG (DT) can participate in meibogenesis, we studied mice whose diet was supplemented with trace amounts of deuterated Chl (2H-Chl) and 13C-labeled triolein (13C-TO), and the products of their biosynthetic transformations were analyzed using LC/MS. We demonstrated that 2H-Chl, but not 13C-TO, could be directly incorporated into meibum. Furthermore, 2H-Chl was esterified into MG-specific ultra long 2H-CE, which were vastly different from plasma CE and 2H-CE. The measured 2H-Chl/Chl and 2H-CE/CE ratios in meibum increased in a time-dependent manner reaching ∼5% and ∼1.2 %, respectively. The 2H-Chl/2H-CE ratio was about 3.5x higher than that for endogenous unlabeled Chl and CE, indicating accumulation of 2H-Chl in meibum. The elongation pattern of Meibomian 2H-CE closely replicated that of unlabeled CE. On the other hand, 13C-TO was not detected in any of the ML samples as an intact lipid or its metabolized/hydrolyzed products. We conclude that DC can be directly esterified into MG-specific CE, while DT undergo extensive catabolic transformations before reaching MG. These findings demonstrate that DC can have a direct impact on MG and ocular surface lipid homeostasis and pathophysiology.

Depletion of Cholesteryl Esters Causes Meibomian Gland Dysfunction-Like Symptoms in a Soat1-Null Mouse Model.

Previous studies on ablation of several key genes of meibogenesis related to fatty acid elongation, omega oxidation, and esterification into wax esters have demonstrated that inactivation of any of them led to predicted changes in the meibum lipid profiles and caused severe abnormalities in the ocular surface and Meibomian gland (MG) physiology and morphology. In this study, we evaluated the effects of Soat1 ablation that were expected to cause depletion of the second largest class of Meibomian lipids (ML)-cholesteryl esters (CE)-in a mouse model. ML of the Soat1-null mice were examined using liquid chromatography high-resolution mass spectrometry and compared with those of Soat1+/- and wild-type mice. Complete suppression of CE biosynthesis and simultaneous accumulation of free cholesterol (Chl) were observed in Soat1-null mice, while Soat1+/- mutants had normal Chl and CE profiles. The total arrest of the CE biosynthesis in response to Soat1 ablation transformed Chl into the dominant lipid in meibum accounting for at least 30% of all ML. The Soat1-null mice had clear manifestations of dry eye and MG dysfunction. Enrichment of meibum with Chl and depletion of CE caused plugging of MG orifices, increased meibum rigidity and melting temperature, and led to a massive accumulation of lipid deposits around the eyes of Soat1-null mice. These findings illustrate the role of Soat1/SOAT1 in the lipid homeostasis and pathophysiology of MG.

Physiological effects of inactivation and the roles of Elovl3/ELOVL3 in maintaining ocular homeostasis.

Recently, elongase of very long chain fatty acids-3 (ELOVL3) was demonstrated to play a pivotal role in physiology and biochemistry of the ocular surface by maintaining a proper balance in the lipid composition of meibum. The goal of this study was to further investigate the effects of ELOVL3 ablation in homozygous Elovl3-knockout mice (E3hom) in comparison with age and sex matched wild-type controls (E3wt). Slit lamp examination of the ocular surface of mice, and histological examination of their ocular tissues, highlighted a severe negative impact of Elovl3 inactivating mutation on the Meibomian glands (MG) and conjunctiva of mice. MG transcriptomes of the E3hom and E3wt mice were assessed and revealed a range of up- and downregulated genes related to lipid biosynthesis, inflammation, and stress response, compared with E3wt mice. Heat stage polarized light microscopy was used to assess melting characteristics of normal and abnormal meibum. The loss of Elovl3 led to a 8°C drop in the melting temperature of meibum in E3hom mice, and increased its fluidity. Also noted were the excessive accumulation of lipid material and tears around the eye and severe ocular inflammation, among other abnormalities.

Comprehensive profiling of Asian and Caucasian meibomian gland secretions reveals similar lipidomic signatures regardless of ethnicity.

Meibum-a lipid secretion that is produced by Meibomian glands (MG) in a process termed meibogenesis-plays a critical role in ocular surface physiology. Abnormalities in the chemical composition of meibum were linked to widespread ocular pathologies-dry eye syndrome (DES) and MG dysfunction (MGD). Importantly, in epidemiologic studies the Asian population was shown to be prone to these pathologies more than the Caucasian one, which was tied to differences in their meibomian lipids. However, biochemical data to support these observations and conclusions are limited. To determine if non-DES/non-MGD Asian meibum was significantly different from that of Caucasians, individual samples of meibum collected from ethnic Asian population living in Japan were compared with those of Caucasians living in the USA. These experiments revealed that composition of major lipid classes, such as wax esters (WE), cholesteryl esters (CE), triacylglycerols, (O)-acylated ω-hydroxy fatty acids (OAHFA), cholesteryl sulfate, cholesteryl esters of OAHFA, and diacylated α,ω-dihydroxy fatty alcohols remained invariable in both races, barring a minor (< 10%; p < 0.01) increase in the Asian CE/WE ratio. Considering the natural variability range for most meibomian lipids (app. ± 15% of the Mean), these differences in meibogenesis were deemed to be minimal and unlikely to have a measurable physiological impact.

Delineating a novel metabolic high triglycerides-low waxes syndrome that affects lipid homeostasis in meibomian and sebaceous glands.

Meibomian glands that are embedded in tarsal plates of human eyelids, and sebaceous glands found in the skin, including that of eyelids, are two related types of holocrine glands that produce lipid-rich secretions called meibum and sebum. Pervasive ocular disorders, such as Meibomian gland dysfunction and dry eye, have been linked to changes in the lipid composition of meibum. However, in most described cases the changes were either small, or random, or insufficiently characterized on the molecular level. Here, we present results of comprehensive lipidomic analyses of meibum, tears and sebum of a patient whose secretions were highly abnormal (abnormal meibum, tears and sebum, or AMTS, patient). The lipidomes were characterized on the level of individual lipid species using ultra-high performance liquid chromatography and high resolution mass spectrometry. The major differences between the AMTS patient and normal age- and gender-matched subjects included, among others, severely diminished pools of normal meibomian lipids such as wax esters and cholesteryl esters in meibum and tears, a 2x increase in total cholesteryl esters to wax esters ratio, their skewed molecular profiles, a ~3x increase in free cholesterol to cholesteryl esters ratio, and, most importantly, a 20x to 30x increase in the triglicerides fraction over the norm. Sebum of the AMTS patient was also highly abnormal lacking major wax esters. Notably, the routine blood lipid panel test of the AMTS patient showed no abnormalities. The data imply that the AMTS patient had a severe, previously unreported, metabolic disorder that affected meibogenesis in Meibomian glands and sebogenesis in sebaceous glands. This is, to the best of our knowledge, a first observation of the condition that we have termed High Triglycerides/Low Waxes (HTLW) syndrome.

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.

On the pivotal role of Elovl3/ELOVL3 in meibogenesis and ocular physiology of mice.

The purpose of this study was to examine the role of Elovl3 gene in meibogenesis and the impact of ELOVL3 protein ablation on the physiology of the mouse ocular surface and Meibomian glands (MGs). Elovl3 knockout, ELOVL3-ablated (E3hom) mice and their wild type littermates (E3wt) were studied side by side. E3hom mice had abnormal ocular phenotypes such as delayed eye opening, weeping eyes, crusty eyelids, eyelid edema, highly vascularized cornea and tarsal plates (TPs), slit eye, and increased tearing that resemble symptoms observed in human subjects with various forms of dry eye, MG dysfunction and blepharitis. Lipid profiling of E3hom TPs was conducted using chromatography and mass spectrometry. The analyses revealed that the lipid composition of E3hom TPs was strikingly different from that of their E3wt littermates. The mutation affected major classes of meibomian lipids - cholesteryl esters, wax esters, and cholesteryl esters of (O)-acylated w-hydroxy fatty acids. The studies illuminated the central role of ELOVL3 in producing C21:0-C29:0 fatty acids, including odd-chain and branched ones. Ablation of ELOVL3 leads to selective changes in the lipid composition of meibum, making E3hom mice instrumental in studying the mechanisms of the biosynthesis of meibum and modeling various pathologies of human ocular surface and adnexa.-Butovich, I. A., Wilkerson, A., Bhat, N., McMahon, A., Yuksel, S. On the pivotal role of Elovl3/ELOVL3 in meibogenesis and ocular physiology of mice.

Cyclic Change of Fatty Acid Composition in Meibum During the Menstrual Cycle.

Purpose: To evaluate the fatty acid (FA) composition in the meibum of pre- and postmenopausal women and age-matched men. Methods: This prospective study involved 24 healthy subjects; six premenopausal women in their 30s with a regular menstrual cycle (young-female [yF] group), six postmenopausal women in their 60s (elderly-female [eF] group), and 12 age-matched men (i.e., young-male [yM] and elderly-male [eM] groups, respectively). The menstrual cycle was divided into six phases (phase I-VI). Meibum was obtained from the meibomian gland orifices via a Daviel spoon, and its FA composition was then analyzed via gas chromatography mass spectrometry (GC-MS). Principal component analysis (PCA) was performed on the GC-MS results. Results: The mean FA composition of all subjects was 40% saturated FAs (SFA) and 60% unsaturated FAs (UFAs). The PCA results of all groups indicated two categories (PC1 [77.5%] and PC2 [12.4%]); one consisting of yF-group samples of mainly phase II and III and the other consisting of the yF-group samples of the rest of the cycle, as well as from eF-group, yM-group, and eM-group samples. Each group had a distinctive nature. The FAs that most contributed to PC1 were C14:0, C16:0, and C18:0 in a positive correlation, and C18:1n9 in a negative correlation. Conclusions: FA composition noticeably changes during the menstrual cycle and is somewhat affected by sex and age. The ratio of SFAs (C16:0, C18:0) to mono-UFAs (C18:1n9) in the FA composition might have an impact on the lipid quality of meibum, thus suggesting alteration of its melting temperature and viscosity.

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.