Evaluating the effect of pulse energy on femtosecond laser trabeculotomy (FLT) outflow channels for glaucoma treatment in human cadaver eyes.

BACKGROUND AND OBJECTIVES: Femtosecond laser trabeculotomy (FLT) creates aqueous humor outflow channels through the trabecular meshwork (TM) and is an emerging noninvasive treatment for open-angle glaucoma. The purpose of this study is to investigate the effect of pulse energy on outflow channel creation during FLT. MATERIALS AND METHODS: An FLT laser (ViaLase Inc.) was used to create outflow channels through the TM (500 µm wide by 200 µm high) in human cadaver eyes using pulse energies of 10, 15, and 20 µJ. Following treatment, tissues were fixed in 4% paraformaldehyde. The channels were imaged using optical coherence tomography (OCT) and assessed as full thickness, partial thickness, or not observable. RESULTS: Pulse energies of 15 and 20 µJ had a 100% success rate in creating full-thickness FLT channels as imaged by OCT. A pulse energy of 10 µJ resulted in no channels (n = 6), a partial-thickness channel (n = 2), and a full-thickness FLT channel (n = 2). There was a statistically significant difference in cutting widths between the 10 and 15 µJ groups (p < 0.0001), as well as between the 10 and 20 µJ groups (p < 0.0001). However, there was no statistically significant difference between the 15 and 20 µJ groups (p = 0.416). CONCLUSIONS: Fifteen microjoules is an adequate pulse energy to reliably create aqueous humor outflow channels during FLT in human cadaver eyes. OCT is a valuable tool when evaluating FLT.

The Yin and Yang of non-immune and immune responses in meibomian gland dysfunction.

Meibomian gland dysfunction (MGD) is a leading cause of dry eye disease and one of the most common ophthalmic conditions encountered in eye clinics worldwide. These holocrine glands are situated in the eyelid, where they produce specialized lipids, or meibum, needed to lubricate the eye surface and slow tear film evaporation - functions which are critical to preserving high-resolution vision. MGD results in tear instability, rapid tear evaporation, changes in local microflora, and dry eye disease, amongst other pathological entities. While studies identifying the mechanisms of MGD have generally focused on gland obstruction, we now know that age is a major risk factor for MGD that is associated with abnormal cell differentiation and renewal. It is also now appreciated that immune-inflammatory disorders, such as certain autoimmune diseases and atopy, may trigger MGD, as demonstrated through a T cell-driven neutrophil response. Here, we independently discuss the underlying roles of gland and immune related factors in MGD, as well as the integration of these two distinct mechanisms into a unified perspective that may aid future studies. From this unique standpoint, we propose a revised model in which glandular dysfunction and immunopathogenic pathways are not primary versus secondary contributors in MGD, but are fluid, interactive, and dynamic, which we likened to the Yin and Yang of MGD.

Modeling meibum secretion: Alternatives for obstructive Meibomian Gland Dysfunction (MGD).

PURPOSE: While changes in meibum quality are correlated with severity of meibomian gland dysfunction (MGD) and dry eye disease, little is known regarding the mechanics of meibum secretion. The purpose of this study was to develop a finite element model of meibum secretion and evaluate the effect of various factors that might impact meibum delivery to the ocular surface. METHODS: A finite element analysis in COMSOL 6.0 was used to simulate the flow of meibum within the gland's terminal excretory duct. Historical normal human meibum rheology data taken over the meibum melting range from fluid (35-40 °C) to solid (25-30 °C) were then used to calculate the minimum yield stress and plastic viscosity of meibum. The effects of meibum melting state, eyelid pressure and terminal duct diameter on meibum flow rates were then systematically investigated. RESULTS: The melting state of meibum from liquid to solid was associated with an increase in the minimum yield stress and plastic viscosity that caused an exponential decrease in meibum flow. Modeling also established that there was a linear correlation between meibum flow rate and eyelid pressure needed to express meibum and the 4th power of the terminal duct radius. CONCLUSIONS: Our results suggest that changes in the melting state of meibum from fluid to solid, as well as changes in the radius of the terminal excretory duct and the force exerted by the eyelid can lead to dramatic decreases in the flow of meibum. Together these findings suggest alternative mechanisms for meibomian gland obstruction.

Meibomian gland stem/progenitor cells: The hunt for gland renewal.

Meibomian glands (MGs) secrete lipid (meibum) onto the ocular surface to form the outermost layer of the tear film. Proper meibum secretion is essential for stabilizing the tear film, reducing aqueous tear evaporation, and maintaining the homeostasis of the ocular surface. Atrophy of MG as occurs with aging, leads to reduction of meibum secretion, loss of ocular surface homeostasis and evaporative dry eye disease (EDED). Since MGs are holocrine glands, secretion of meibum requires continuous self-renewal of lipid-secreting acinar meibocytes by stem/progenitor cells, whose proliferative potential is dramatically reduced with age leading to MG atrophy and an age-related meibomian gland dysfunction (ARMGD). Understanding the cellular and molecular mechanisms regulating meibocyte stem/progenitor cell maintenance and renewal may provide novel approaches to regenerating MG and treating EDED. Towards that end, recent label retaining cell and lineage-tracing experiments as well as knock-out transgenic mouse studies have begun to identify the location and identities of meibocyte progenitor cells and potential growth and transcription factors that may regulate meibocyte renewal. In addition, recent reports have shown that ARMGD may be reversed by novel therapeutics in mice. Herein, we discuss our current understanding of meibocyte stem/progenitor cells and the hunt for gland renewal.

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.

Immuno Tomography (IT) and Imaging Mass Cytometry (IMC) for constructing spatially resolved, multiplexed 3D IMC data sets.

PURPOSE: We have previously used Immuno Tomography (IT) to identify label-retaining stem cell populations in the cornea and meibomian gland. While this method provides the unique ability to quantify stem cell populations comprised of 1-4 cells, the number of antigens that can be sequentially used to characterize these unique cells is limited by antigen stability after antibody stripping and re-probing. To address this deficiency, we have evaluated the capability of Imaging Mass Cytometry™ (IMC™) to generate multiplexed images using metal-conjugated antibodies to label IT plastic sections and generate 3-dimensional IMC data sets (3D IMC). METHODS: K5-H2B-GFP mice, 56 days after doxycycline chase, were sacrificed and eyelid tissue processed for IT. A total of 400 serial, plastic sections, 2 µm thick, were then probed using metal-tagged antibodies specific for sox 9, collagen type I, E-cadherin, Ki67, GFP, αSMA, vimentin, and DNA intercalator. Multiplexed images were then generated using an Imaging Mass Cytometry system (Fluidigm®), and 3D reconstructions were assembled. RESULTS: All 8 metal-labeled tags were detected and their images were successfully assembled into 3D IMC data sets. GFP-labeled nuclei were identified within the meibomian glands in comparable numbers to those previously reported for slow-cycling meibomian gland stem cells. CONCLUSIONS: These findings demonstrate that IMC can be used on plastic sections to generate multiplexed, 3D data sets that can be reconstructed to show the spatial localization of meibomian gland stem cells. We propose that 3D IMC might prove valuable in more fully characterizing stem cell populations in different tissues.

Femtosecond Laser Trabeculotomy in Perfused Human Cadaver Anterior Segments: A Novel, Noninvasive Approach to Glaucoma Treatment.

Purpose: The purpose of this study was to investigate femtosecond laser trabeculotomy (FLT) in a clinically relevant manner (i.e., delivering the surgical laser beam through the cornea of the intact, human anterior segment to create channels from the anterior chamber into the Schlemm's canal) and to investigate the effect of this treatment on intraocular pressure in perfused human anterior segments. Methods: Perfused human anterior segments (15 eyes) received either FLT treatment (n = 8) or a sham-treatment (n = 7). Intraocular pressure (IOP) in the perfused samples was recorded before and after treatment. Spectral domain optical coherence tomography, second harmonic generation imaging, and transmission electron microscopy were used to investigate the FLT channels. Results: The FLT group (n = 7, 1 eye excluded) had a statistically significant reduction in mean IOP of 20.2% from baseline after treatment (5.06 ± 1.46 mm Hg to 4.04 ± 1.63 mm Hg; P < 0.0005), whereas the control group (n = 7) remained statistically unchanged (7.72 ± 3.45 mm Hg to 7.78 ± 3.51 mm Hg; P < 0.71). Imaging confirmed that the channels traversed the entire trabecular meshwork into the Schlemm's canal. Conclusions: This study has provided the first direct evidence supporting the feasibility of clinically applicable, noninvasive femtosecond laser trabeculotomy for the treatment of glaucoma. Various imaging modalities revealed minimal collateral damage to adjacent issues. Translational Relevance: This work demonstrates noninvasive femtosecond laser trabeculotomy in a laboratory setting that is clinically relevant.

The Efficacy of Clinical Tests to Diagnose Evaporative Dry Eye Disease Related to Meibomian Gland Dysfunction.

OBJECTIVES: To determine the efficacy of widely available subtype clinical tests to characterize evaporative dry eye disease (EDED) related to meibomian gland dysfunction (MGD) compared to normal and to validate those clinical cut points in an independent sample. METHODS: A diagnostic accuracy study (52 subjects), an investigator-masked study, was followed by a larger independent sample (364 subjects) analysis to confirm efficacy in normal and EDED subjects. All subjects were 18 years of age and older and were classified using a battery of clinical tests for dry eye that included symptoms, tear meniscus height, tear stability, ocular staining, evaporative-specific tests, and the Schirmer I test. RESULTS: Normal (nondry eye; n = 26) and EDED (n = 26) subjects completed the efficacy study. The global tests of tear breakup time, staining, and symptoms all produced AUCs ≥ 0.70, representing acceptable discrimination. EDED-specific tests of eyelid marginal signs, gland secretion quality, and gland loss did not demonstrate acceptable test efficacy or differences between normal and EDED subjects. In a larger, independent sample of normal and EDED subjects, gland secretion quality and eyelid marginal score achieved acceptable diagnostic levels: AUCs of 0.789 (CI: 0.734-0.844) and 0.729 (CI: 0.648-0.810), respectively, but not lipid interferometry grade or lower eyelid gland dropout estimated using meiboscopy. CONCLUSIONS: Meibomian gland secretion quality is an efficient and useful functional indicator in EDED and should be incorporated into core outcome sets for this dry eye subtype.

Ascorbic acid specifically reduces the misclassification of nonirritating reactive chemicals in the OptiSafe™ macromolecular eye irritation test.

Recently, we showed that the addition of physiological concentrations of ascorbic acid, a tear antioxidant, to the OptiSafe™ macromolecular eye irritation test reduced the false-positive (FP) rate for chemicals that had reactive chemistries, leading to the formation of reactive oxygen species (ROS) and molecular crosslinking. The purpose of the current study was to 1) increase the number of chemicals tested to comprehensibly determine whether the antioxidant-associated reduction in OD is specific to FP chemicals associated with ROS chemistries and 2) determine whether the addition of antioxidants interferes with the detection of true positive (TP) and true negative (TN) ocular irritants. We report that when ascorbic acid is added to the test reagents, retesting of FP chemicals with reactive chemistries show significantly reduced OD values (P < 0.05). Importantly, ascorbic acid had no significant effect on the OD values of TP or TN chemicals regardless of chemical reactivity. These findings suggest that supplementation of ascorbic acid in alternative ocular irritation tests may help improve the detection of TN for those commonly misclassified reactive chemicals.

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.

Response to Letter to Editor "Comments on 'Cell regulation of collagen fibril macrostructure during corneal morphogenesis' by Koudouna et al."

STATEMENT OF SIGNIFICANCE:  .

Intraocular Pressure Reduction by Femtosecond Laser Created Trabecular Channels in Perfused Human Anterior Segments.

Purpose: This study investigated the initial feasibility of using femtosecond laser trabeculotomy (FLT) to create open channels through the trabecular meshwork into Schlemm's canal to lower intraocular pressure (IOP) in a perfused anterior segment model. Methods: Human anterior segments (12 eyes) were assigned to either treatment (n = 6) or sham treatment (n = 6) groups. Both groups were perfused until a baseline IOP was recorded upon which a direct FLT treatment or a sham treatment was administered. IOP was recorded before and after the treatment. Spectral domain optical coherence tomography and second harmonic generation imaging we used to investigate the FLT channels. Results: In the FLT group, there was a significant mean decrease in the IOP of 22% compared with the pre-FLT IOP (7.13 ± 2.95 mm Hg to 5.34 ± 1.62 mm Hg; P < 0.05). In the control group, the post-sham IOP remained relatively unchanged compared with the pre-sham IOP (6.39 ± 3.69 mm Hg to 6.67 ± 4.12 mm Hg). Conclusions: The results of this study indicate that FLT treatment can significantly decrease the IOP in a perfusion model and may provide a potential noninvasive treatment option for primary open angle glaucoma. Translational Relevance: Investigating the use of femtosecond lasers for photodisrupting the trabecular meshwork can lead to a clinically relevant alternative to current glaucoma procedures.

Modeling the antioxidant properties of the eye reduces the false-positive rate of a nonanimal eye irritation test (OptiSafe).

We recently identified a group of chemicals that are misclassified by most, if not all, in vitro alternative ocular irritation tests, suggesting that nonanimal tests may not fully model the ocular environment in which these chemicals interact. To address this, we evaluated the composition of tears, the first defense against foreign substances, and identified the presence of antioxidants that could detoxify reactive chemicals that otherwise may be falsely identified as irritants in alternative irritation tests. In this study, we evaluated the effects of tear antioxidants on the ocular irritation scoring of commonly overclassified chemicals (false positives) using the OptiSafe™ ocular irritation test. Six tear-related antioxidants were individually added to the OptiSafe formulation, and the effects on test outcome were determined. Ascorbic acid, the most abundant water-soluble antioxidant in tears, specifically reduced the OptiSafe false-positive rate. Titration curves showed that this reduction occurs at in vivo concentrations and is specific to chemicals identified either as producing reactive oxygen species or as crosslinkers. Importantly, the addition of tear antioxidants did not impact the detection of true negatives, true positives, or other false positives unassociated with reactive oxygen species or crosslinking. These results suggest that the addition of tear antioxidants to in vitro alternative test systems may substantially reduce the false-positive rate and improve ocular irritant detection.

A novel transillumination meibography device for in vivo imaging of mouse meibomian glands.

PURPOSE: While mouse models of dry eye disease (DED) have been developed, studies evaluating the role of the meibomian glands limited by the inability to temporally document changes. In this report we describe the development of a novel mouse transillumination meibography device and assess the ability of this device to detect age-related changes in the meibomian glands of young and old mice. METHODS: The mouse meibography device was comprised of a 3 mm wide right angle prism attached to broad spectrum light source by an optical fiber. Eyelids were then pulled over the prism using double tooth forceps and imaged using a stereomicroscope and low light level camera. Meibomian glands from four young and four old male, BALB/c mice were then imaged and analyzed using ImageJ. RESULTS: In young mice, meibography documented the presence of 7-8 meibomian glands appearing as black and distinct eyelid structures with the length shorter in the lower eyelid compared to the upper eyelids. Eyelids of old mice showed apparent dropout of meibomian glands along with smaller and more irregularly shaped acini. The mean acini area of one meibomian gland was 0.088 ± 0.025 mm2 in young mice and 0.080 ± 0.020 mm2 in old mice (p = 0.564), but the Meibomian gland density was significantly lower in older mice (41.7 ± 6.4%, 27.3 ± 4.2%) (p = 0.021). CONCLUSION: We have developed an in vivo meibography device that may prove useful in sequentially documenting changes during development of meibomian gland dysfunction and following treatment.

Same-chemical comparison of nonanimal eye irritation test methods: Bovine corneal opacity and permeability, EpiOcular™, isolated chicken eye, ocular Irritection®, OptiSafe™, and short time exposure.

The testing and classification of chemicals to determine adverse ocular effects are routinely conducted to ensure that materials are appropriately classified, labeled, and meet regulatory and safety guidelines. We have performed a same-chemical analysis using publicly available validation study results and compared the performance between tests for the same chemicals. To normalize for chemical selection, we matched chemicals tested by pairs of tests so that each matched set compared performance for the exact same chemicals. Same-chemical accuracy comparisons demonstrate a chemical selection effect that results in a wide range of overlapping false-positive (FP) rates and accuracies for all test methods. In addition, the analysis suggests that a tiered-testing strategy with specific combinations of tests can reduce the FP rate for some combinations. However, reductions in the FP rates were typically accompanied by an increase in the false-negative rates, resulting in minimal advantage in terms of accuracy. In addition, actual improvements in the FP rate after retesting positives with a second test are not as good as the theoretical improvements because some chemicals and functional groups appear to be broadly misclassified by all test methods, which, to the extent the tests make the same-chemical misclassifications, reduces the advantage of using tiered-testing strategies.

Nonlinear optical crosslinking (NLO CXL) for correcting refractive errors.

Ultraviolet A (UVA) light-based photoactivation of riboflavin (Rf) to induce corneal crosslinking (CXL) and mechanical stiffening is now a well-known treatment for corneal ectasia and Keratoconus that is being used in a topographically guided photorefractive intrastromal CXL (PiXL) procedure to treat low degrees of refractive errors. Alternative approaches for non-invasive treatment of refractive errors have also been proposed that use femtosecond lasers (FS) that provide much faster, more precise, and safer results than UVA CXL. One such treatment, nonlinear optical crosslinking (NLO CXL), has been able to replicate the effects of UVA CXL, while producing a smaller area of cellular damage and requiring a shorter procedure time. Unlike UVA CXL, the treatment volume of NLO CXL only occurs within the focal volume of the laser, which can be placed at any depth and scanned into any pattern for true topographically guided refractive correction. This review presents our experience with using FS lasers to photoactivate Rf and perform highly controlled corneal CXL that leads to mechanical stiffening and changes in corneal shape.

Recapitulation of normal collagen architecture in embryonic wounded corneas.

Wound healing is characterized by cell and extracellular matrix changes mediating cell migration, fibrosis, remodeling and regeneration. We previously demonstrated that chick fetal wound healing shows a regenerative phenotype regarding the cellular and molecular organization of the cornea. However, the chick corneal stromal structure is remarkably complex in the collagen fiber/lamellar organization, involving branching and anastomosing of collagen bundles. It is unknown whether the chick fetal wound healing is capable of recapitulating this developmentally regulated organization pattern. The purpose of this study was to examine the three-dimensional collagen architecture of wounded embryonic corneas, whilst identifying temporal and spatial changes in collagen organization during wound healing. Linear corneal wounds that traversed the epithelial layer, Bowman´s layer, and anterior stroma were generated in chick corneas on embryonic day 7. Irregular thin collagen fibers are present in the wounded cornea during the early phases of wound healing. As wound healing progresses, the collagen organization dramatically changes, acquiring an orthogonal arrangement. Fourier transform analysis affirmed this observation and revealed that adjacent collagen lamellae display an angular displacement progressing from the epithelium layer towards the endothelium. These data indicate that the collagen organization of the wounded embryonic cornea recapitulate the native macrostructure.

Enhanced Transepithelial Riboflavin Delivery Using Femtosecond Laser-Machined Epithelial Microchannels.

Purpose: This study describes a femtosecond laser (FS) approach to machine corneal epithelial microchannels for enhancing riboflavin (Rf) penetration into the cornea prior to corneal crosslinking (CXL). Methods: Using a 1030-nm FS laser with 5- to 10-µJ pulse energy, the corneal epithelium of slaughterhouse rabbit eyes was machined to create 2-µm-diameter by 25-µm-long microchannels at a density of 100 or 400 channels/mm2. Rf penetration through the microchannels was then determined by applying 1% Rf in phosphate-buffered saline for 30 minutes followed by removal of the cornea and extraction from the central stromal button. Stromal Rf concentrations were then compared to those obtained using standard epithelial debridement or 0.01% benzalkonium chloride (BAK) to disrupt the epithelial barrier. Results: Microchannels formed using a 5-µJ/pulse at a density of 400 channels/mm2 achieved a stromal Rf concentration that was 50% of that achieved by removal of the corneal epithelium and imbibing with 1% Rf. Stromal Rf levels were also equal to that of debrided corneas soaked with 0.5% Rf, threefold higher than those soaked with 0.1% Rf, and twofold higher than corneas soaked in BAK without epithelial debridement. Organ culture of treated corneas showed a normal corneal epithelium following FS machining while BAK-treated corneas showed extensive epithelial and stromal damage at 24 hours posttreatment. Conclusions: FS corneal epithelial machining can be used to enhance penetration of Rf into the stroma for corneal CXL. Translational Relevance: The creation of epithelial microchannels allows for stromal Rf concentrations high enough to perform true transepithelial crosslinking.

Origin and Lineage Plasticity of Endogenous Lacrimal Gland Epithelial Stem/Progenitor Cells.

The lacrimal gland (LG) is an exocrine organ responsible for the secretion of aqueous tear film. Regenerative and stem cell therapies that target LG repair are coming to the fore, although our understanding of LG cell lineage hierarchy is still incomplete. We utilize the analysis of label-retaining cells (LRCs) and genetic lineage tracing to define LG cell lineage hierarchy. Our study suggests that embryonic LG contains unique long-lived multipotent stem cells that give rise to all postnatal epithelial cell types. Following birth, lineages become established and the fate of progenitor cell descendants becomes restricted. However, some cell lineages retain plasticity after maturation and can trans-differentiate into other cell types upon injury. The demonstration that the LG contains progenitor cells with different levels of plasticity has profound implications for our understanding of LG gland function in homeostasis and disease and will be helpful for developing stem cell-based therapies in the future.

Eicosapentaenoic acid (EPA) activates PPARγ signaling leading to cell cycle exit, lipid accumulation, and autophagy in human meibomian gland epithelial cells (hMGEC).

PURPOSE: The purpose of this study was to access the ability of the natural PPAR agonist, eicosapentaenoic acid (EPA), to activate PPAR gamma (γ) signaling leading to meibocyte differentiation in human meibomian gland epithelial cell (hMGEC). METHODS: HMGEC were exposed to EPA, alone and in combination with the specific PPARγ antagonist, T0070907, to selectively block PPARγ signaling. Expression of PPARγ response genes were evaluated by qPCR. Effect on cell cycle was evaluated using Ki-67 labelling and western blots. During differentiation, autophagy was monitored using the Autophagy Tandem Sensor (ATS) and LysoTracker. Lipid accumulation was characterized by Stimulated Raman Scattering microscopy (SRS) and neutral lipid staining in combination with ER-Tracker, LysoTracker, and ATS. Autophagy was also investigated using western blotting. Seahorse XF analysis was performed to monitor mitochondrial function. RESULTS: EPA specifically upregulated expression of genes related to lipid synthesis and induced cell cycle exit through reduced cyclin D1 expression and increased p21 and p27 expression. EPA also induced accumulation of lipid droplets in a time and dose dependent manner (P < 0.05) by specific PPARγ signaling. Lipid analysis identified both de novo synthesis and extracellular transport of lipid to form lipid droplets that were localized to the ER. PPARγ signaling also induced activation of AMPK-ULK1 signaling and autophagy, while inhibition of autophagy induced mitochondrial crisis with no effect on lipid accumulation. CONCLUSIONS: EPA induces meibocyte differentiation through PPARγ activation that is characterized by cell cycle exit, de novo and transported lipid accumulation in the ER, and autophagy.