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

PURPOSE: Recent 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. METHODS: iHMGEC 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. RESULTS: Under 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. CONCLUSION: The 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.

Acyl-CoA:wax alcohol acyltransferase 2 modulates the cone visual cycle in mouse retina.

The daylight and color vision of diurnal vertebrates depends on cone photoreceptors. The capability of cones to operate and respond to changes in light brightness even under high illumination is attributed to their fast rate of recovery to the ground photosensitive state. This process requires the rapid replenishing of photoisomerized visual chromophore (11-cis-retinal) to regenerate cone visual pigments. Recently, several gene candidates have been proposed to contribute to the cone-specific retinoid metabolism, including acyl-CoA wax alcohol acyltransferase 2 (AWAT2, aka MFAT). Here, we evaluated the role of AWAT2 in the regeneration of visual chromophore by the phenotypic characterization of Awat2-/- mice. The global absence of AWAT2 enzymatic activity did not affect gross retinal morphology or the rate of visual chromophore regeneration by the canonical RPE65-dependent visual cycle. Analysis of Awat2 expression indicated the presence of the enzyme throughout the murine retina, including the retinal pigment epithelium (RPE) and Müller cells. Electrophysiological recordings revealed reduced maximal rod and cone dark-adapted responses in AWAT2-deficient mice compared to control mice. While rod dark adaptation was not affected by the lack of AWAT2, M-cone dark adaptation both in isolated retina and in vivo was significantly suppressed. Altogether, these results indicate that while AWAT2 is not required for the normal operation of the canonical visual cycle, it is a functional component of the cone-specific visual chromophore regenerative pathway.

Mouse models in studies on the etiology of evaporative dry eye disease.

Evaporative dry eye disease (DED) is a common ocular condition impacting the quality of life of millions of patients worldwide. The etiology of evaporative DED is related to dysfunction of meibomian glands (MGs), resulting in suboptimal yield or lipid composition of secreted meibum. The clinical manifestation of evaporative DED involves mechanical obstruction of the MG orifice and decreased tear film stability that leads to chronic eye irritation, inflammation, and progressive damage to the cornea and surrounding tissue. Despite its high prevalence, evaporative DED remains an unmet medical need. The main obstacle in the development of effective therapeutic strategies against this disease is inadequate knowledge about the complex arrays of lipogenic reactions (meibogenesis) in the MGs and a lack of suitable animal models of the human condition. In this review, we discuss the recent advances in the creation of genetically modified mouse models that recapitulate the phenotype of evaporative DED as well as their impact on our understanding of lipid biosynthesis in MGs and therapeutic strategies targeting meibogenesis.

Expression of Acyl-CoA wax-alcohol acyltransferase 2 (AWAT2) by human and rabbit meibomian glands and meibocytes.

PURPOSE: Previously, we showed that Acyl-CoA wax-alcohol acyltransferase 2 (AWAT2), an essential enzyme required for meibum wax ester synthesis, was not expressed by immortalized human meibomian gland epithelial cells (hMGEC) in culture. To begin to understand the mechanisms controlling AWAT2 expression, we have analyzed its expression in human and rabbit meibomian glands and cultured meibocytes. METHODS: Rabbit meibocyte progenitor cells (rMPC) were first grown in Cnt-BM.1 basal medium (Cellntec) supplemented with rhEGF, FGF10, and ROCK inhibitor (Y-27632 dihydrochloride), and then passed at 70-80% confluency with Accutase. Differentiation of rMPC to meibocytes (rMC) was induced by removal of Y-27632 and addition of 1 mM calcium with and without PPARγ agonists. RNA from the tissue, primary, passaged rMPC and differentiated rMC were obtained for AWAT2 qPCR analysis. Proteins and cells were evaluated for western blotting and neutral lipid synthesis, respectively. For comparison, human meibomian glands were separated for RNA and protein analysis. hMGEC was cultured to collect RNA and protein. RESULTS: Rabbit rMPCs were successfully grown, passaged, and differentiated, showing a significant increase in lipid droplet accumulation. AWAT2 RNA was highly expressed in tissue but showed a -16.9 log2 fold decrease in primary and passaged rMPCs and was not induced by differentiation to rMC. By comparison, human meibomian glands showed high expression of AWAT2, and hMGEC expressed non-detectable levels of AWAT2 transcripts or protein. CONCLUSIONS: AWAT2 expression is lost in cultured rMPC and rMC suggesting that cells in culture do not undergo complete meibocyte differentiation and require yet to be identified culture conditions.

Deficiency in Acyl-CoA:Wax Alcohol Acyltransferase 2 causes evaporative dry eye disease by abolishing biosynthesis of wax esters.

Lipids secreted by the meibomian glands (MGs) of the eyelids are essential to the protection of the eye's surface. An altered meibum composition represents the primary cause of evaporative dry eye disease (DED). Despite the critical importance of the meibum, its biosynthetic pathways and the roles of individual lipid components remain understudied. Here, we report that the genetic deletion of Acyl-CoA:wax alcohol acyltransferase 2 (AWAT2) causes the obstruction of MGs and symptoms of evaporative DED in mice. The lipid composition of the meibum isolated from Awat2-/- mice revealed the absence of wax esters, which was accompanied by a compensatory overproduction of cholesteryl esters. The resulting increased viscosity of meibum led to the dilation of the meibomian ducts, and the progressive degeneration of the MGs. Overall, we provide evidence for the main physiological role of AWAT2 and establish Awat2-/- mice as a model for DED syndrome that can be used in studies on tear film-oriented therapies.