Lee SJ, Kim S-T, Wu J, Cho CS, Jo DH, Chen Y, Dana R, Kim JH, Lee S-M. Corneal lymphangiogenesis in dry eye disease is regulated by substance P/neurokinin-1 receptor system through controlling expression of vascular endothelial growth factor receptor 3. Ocul Surf 2021;22:72-79.Abstract
PURPOSE: To evaluate the role of substance P (SP)/neurokinin-1 receptor (NK1R) system in the regulation of pathologic corneal lymphangiogenesis in dry eye disease (DED). METHODS: Immunocytochemistry, angiogenesis assay, and Western blot analysis of human dermal lymphatic endothelial cells (HDLECs) were conducted to assess the involvement of SP/NK1R system in lymphangiogenesis. DED was induced in wild-type C57BL/6 J mice using controlled-environment chamber without scopolamine. Immunohistochemistry, corneal fluorescein staining, and phenol red thread test were used to evaluate the effect of SP signaling blockade in the corneal lymphangiogenesis. The expression of lymphangiogenic factors in the corneal and conjunctival tissues of DED mouse model was quantified by real-time polymerase chain reaction. RESULTS: NK1R expression and pro-lymphangiogenic property of SP/NK1R system in HDLECs were confirmed by Western blot analysis and angiogenesis assay. Blockade of SP signaling with L733,060, an antagonist of NK1R, or NK1R-targeted siRNA significantly inhibited lymphangiogenesis and expression of vascular endothelial growth factor (VEGF) receptor 3 stimulated by SP in HDLECs. NK1R antagonist also suppressed pathological corneal lymphangiogenesis and ameliorated the clinical signs of dry eye in vivo. Furthermore, NK1R antagonist effectively suppressed the lymphangiogenic factors, including VEGF-C, VEGF-D, and VEGF receptor 3 in the corneal and conjunctival tissues of DED. CONCLUSIONS: SP/NK1R system promotes lymphangiogenesis in vitro and NK1R antagonism suppresses pathologic corneal lymphangiogenesis in DED in vivo.
Lee R, Khoueir Z, Tsikata E, Chodosh J, Dohlman CH, Chen TC. Long-term Visual Outcomes and Complications of Boston Keratoprosthesis Type II Implantation. Ophthalmology 2017;124(1):27-35.Abstract

PURPOSE: To report the long-term visual outcomes and complications after Boston keratoprosthesis type II implantation in the largest single-center case series with the longest average follow-up. DESIGN: Retrospective review of consecutive clinical case series. PARTICIPANTS: Between January 1992 and April 2015 at the Massachusetts Eye and Ear Infirmary, 48 eyes of 44 patients had keratoprosthesis type II implanted by 2 surgeons (C.H.D. and J.C.). METHODS: For each eye, data were collected and analyzed on the preoperative characteristics, intraoperative procedures, and postoperative course. MAIN OUTCOME MEASURES: Visual acuity outcomes, postoperative complications, and device retention. RESULTS: The most common indications for surgery were Stevens-Johnson syndrome in 41.7% (20 of 48 eyes) and mucous membrane pemphigoid in 41.7% (20 of 48 eyes). Mean follow-up duration was 70.2 months (standard deviation, 61.8 months; median, 52 months; range, 6 months to 19.8 years). Almost all patients (95.8%, 46 of 48 eyes) had a preoperative visual acuity of 20/200 or worse. Postoperative visual acuity improved to 20/200 or better in 37.5% (18 of 48 eyes) and to 20/100 or better in 33.3% (16 of 48 eyes) at the last follow-up visit. The most common postoperative complication was retroprosthetic membrane formation in over half (60.4%, 29 of 48 eyes). The most pressing postoperative complication was glaucoma onset or progression in about a third. Preexisting glaucoma was present in 72.9% (35 of 48 eyes). Glaucoma progressed in 27.1% (13 of 48 eyes) and was newly diagnosed in 8.3% (4 of 48 eyes) after surgery. Other postoperative complications were tarsorrhaphy revision in 52.1% (25 of 48 eyes), retinal detachment in 18.8% (9 of 48 eyes), infectious endophthalmitis in 6.3% (3 of 48 eyes), and choroidal detachment or hemorrhage in 8.3% (4 of 48 eyes). Half of eyes retained their initial keratoprosthesis at the last follow-up (50.0%, 24 of 48 eyes). CONCLUSIONS: The Boston keratoprosthesis type II is a viable option to salvage vision in patients with poor prognosis for other corneal procedures. Retroprosthetic membranes, keratoprosthesis retention, and glaucoma are major challenges in the postoperative period; however, the keratoprosthesis can still provide improved vision in a select group of patients.

Lei F, Cui N, Zhou C, Chodosh J, Vavvas DG, Paschalis EI. Reply to Green and Hume: Nonmicroglia peripheral immune effects of short-term CSF1R inhibition with PLX5622. Proc Natl Acad Sci U S A 2021;118(4)
Li D, Hodges RR, Jiao J, Carozza RB, Shatos MA, Chiang N, Serhan CN, Dartt DA. Resolvin D1 and aspirin-triggered resolvin D1 regulate histamine-stimulated conjunctival goblet cell secretion. Mucosal Immunol 2013;6(6):1119-30.Abstract
Resolution of inflammation is an active process mediated by pro-resolution lipid mediators. As resolvin (Rv) D1 is produced in the cornea, pro-resolution mediators could be effective in regulating inflammatory responses to histamine in allergic conjunctivitis. Two key mediators of resolution are the D-series resolvins RvD1 or aspirin-triggered RvD1 (AT-RvD1). We used cultured conjunctival goblet cells to determine whether histamine actions can be terminated during allergic responses. We found cross-talk between two types of G protein-coupled receptors (GPRs), as RvD1 interacts with its receptor GPR32 to block histamine-stimulated H1 receptor increases in intracellular [Ca(2+)] ([Ca(2+)]i) preventing H1 receptor-mediated responses. In human and rat conjunctival goblet cells, RvD1 and AT-RvD1 each block histamine-stimulated secretion by preventing its increase in [Ca(2+)]i and activation of extracellular regulated-protein kinase (ERK)1/2. We suggest that D-series resolvins regulate histamine responses in the eye and offer new treatment approaches for allergic conjunctivitis or other histamine-dependent pathologies.
Li D, Shatos MA, Hodges RR, Dartt DA. Role of PKCα activation of Src, PI-3K/AKT, and ERK in EGF-stimulated proliferation of rat and human conjunctival goblet cells. Invest Ophthalmol Vis Sci 2013;54(8):5661-74.Abstract
PURPOSE: To determine the order and components of the signaling pathway utilized by epidermal growth factor (EGF) to stimulate conjunctival goblet cell proliferation. METHODS: Goblet cells from rat bulbar and forniceal conjunctiva and human bulbar conjunctiva were grown in organ culture. Goblet cells (GCs) were serum starved for 24 hours and preincubated with inhibitors for 30 minutes or small interfering RNA (siRNA) for 48 hours prior to addition of EGF. Proliferation was then measured or Western blot analysis was performed using antibodies against phosphorylated protein kinase B (AKT), extracellular signal-regulated kinase 1/2 (ERK1/2), or the non-receptor tyrosine kinase Src. Rat GCs were also incubated with adenoviruses expressing dominant negative protein kinase Cα (DNPKCα) or constitutively activated protein kinase Cα (myrPKCα), and activation of AKT and ERK1/2 was determined by Western blot analysis. RESULTS: Inhibitors of phosphoinositol-3 kinase (PI-3K)/AKT pathway blocked EGF-stimulated ERK1/2 activation and GC proliferation. Inhibitors of EGF-stimulated ERK1/2 activity did not inhibit AKT activation but blocked proliferation. DNPKCα blocked EGF-stimulated activation of AKT and ERK1/2 while myrPKCα increased activation of these kinases. Inhibitors of PI-3K, ERK1/2, and protein kinase C (PKC) blocked myrPKCα-stimulated GC proliferation. EGF and myrPKCα increased phosphorylation of Src, and inhibition of Src with the chemical inhibitor PP1 or siRNA inhibited EGF-stimulated GC proliferation. CONCLUSIONS: We found that EGF activates a major pathway to stimulate goblet cell proliferation. This pathway consists of induction of phospholipase C (PLC)γ to activate PKCα. Active PKCα phosphorylates Src to induce PI-3K to phosphorylate AKT that subsequently activates the ERK1/2 cascade to stimulate goblet cell proliferation.
Li M, Mittal SK, Foulsham W, Amouzegar A, Sahu SK, Chauhan SK. Mast cells contribute to the induction of ocular mucosal alloimmunity. Am J Transplant 2019;19(3):662-673.Abstract
Beyond their historical role as the effector cells in allergic disorders, mast cells have been implicated in regulating both innate and adaptive immune responses. Possessing considerable functional plasticity, mast cells are abundant at mucosal surfaces, where the host and external environments interface. The purpose of this study was to evaluate the contribution of mast cells to allograft rejection at the ocular surface. Using a well-characterized murine model of corneal transplantation, we report that mast cells promote allosensitization. Our data show mast cell frequencies and activation are increased following transplantation. We demonstrate that mast cell inhibition (a) limits the infiltration of inflammatory cells and APC maturation at the graft site; (b) reduces allosensitization and the generation of Th1 cells in draining lymphoid tissues; (c) decreases graft infiltration of alloimmune-inflammatory cells; and (d) prolongs allograft survival. Our data demonstrate a novel function of mast cells in promoting allosensitization at the ocular surface.
Li D, Jiao J, Shatos MA, Hodges RR, Dartt DA. Effect of VIP on intracellular [Ca2+], extracellular regulated kinase 1/2, and secretion in cultured rat conjunctival goblet cells. Invest Ophthalmol Vis Sci 2013;54(4):2872-84.Abstract
PURPOSE: To determine the intracellular signaling pathways that vasoactive intestinal peptide (VIP) uses to stimulate high molecular weight glycoconjugate secretion from cultured rat conjunctival goblet cells. METHODS: Goblet cells from rat bulbar and forniceal conjunctiva were grown in organ culture. Presence and localization of VIP receptors (VPAC1 and 2) were determined by RT-PCR, immunofluorescence microscopy and Western blot analysis. Intracellular [Ca(2+)] ([Ca(2+)]i) was measured using fura-2. Extracellular signal-regulated kinase (ERK)-1/2 activity was determined by Western blot analysis. High molecular weight glycoconjugate secretion was measured with an enzyme-linked lectin assay on cultured goblet cells that were serum-starved for 2 hours before stimulation with VIP, VPAC1-, or VPAC2-specific agonists. Inhibitors were added 30 minutes prior to VIP. Activation of epidermal growth factor receptor (EGFR) was measured by immunoprecipitation using an antibody against pTyr followed by Western blot analysis with an antibody against EGFR. RESULTS: Both VIP receptors were present in rat conjunctiva and cultured goblet cells. VIP- and VPAC-specific agonists increased [Ca(2+)]i and secretion in a concentration-dependent manner. VIP also increased ERK1/2 activity, VIP-stimulated increase in [Ca(2+)]i. Secretion, but not ERK1/2 activity, was inhibited by the protein kinase A inhibitor, H89. VIP-stimulated secretion was inhibited by siRNA for ERK2 but not by siRNA for EGFR. VIP did not increase the phosphorylation of the EGFR. CONCLUSIONS: In conclusion, in cultured rat conjunctival goblet cells, VPAC1 and 2 receptors are functional. VIP stimulates a cAMP-dependent increase in [Ca(2+)]i and glycoconjugate secretion, but not ERK1/2 activation. VIP does not activate with EGFR.
Li D, Carozza RB, Shatos MA, Hodges RR, Dartt DA. Effect of histamine on Ca(2+)-dependent signaling pathways in rat conjunctival goblet cells. Invest Ophthalmol Vis Sci 2012;53(11):6928-38.Abstract
PURPOSE: The purpose of this study was to determine the Ca(2+)-dependent cellular signaling pathways used by histamine to stimulate conjunctival goblet cell secretion. METHODS: Cultured rat goblet cells were grown in RPMI 1640. Goblet cell secretion of high molecular weight glycoconjugates was measured by an enzyme-linked lectin assay. Intracellular [Ca(2+)] ([Ca(2+)](i)) was measured by loading cultured cells with the Ca(2+) sensitive dye fura-2. The level of [Ca(2+)](i) was measured using fluorescence microscopy. Extracellular regulated kinase (ERK) 2 was depleted using small interfering RNA (siRNA). RESULTS: Histamine-stimulated conjunctival goblet cell secretion of high molecular weight glycoproteins was blocked by removal of extracellular Ca(2+) and depletion of ERK2 by siRNA. Histamine increase in [Ca(2+)](i) was desensitized by repeated addition of agonist and blocked by a phospholipase C antagonist. Histamine at higher doses increased [Ca(2+)](i) by stimulating influx of extracellular Ca(2+), but at a lower dose released Ca(2+) from intracellular stores. Activation of each histamine receptor subtype (H(1)-H(4)) increased [Ca(2+)](i) and histamine stimulation was blocked by antagonists of each receptor subtype. The H(2) receptor subtype increase in [Ca(2+)](i) was cAMP dependent. CONCLUSIONS: We conclude that histamine activates phospholipase C to release intracellular Ca(2+) that induces the influx of extracellular Ca(2+) and activates ERK1/2 to stimulate conjunctival goblet cell mucous secretion, and that activation of all four histamine receptor subtypes can increase [Ca(2+)](i).
Lienert JP, Tarko L, Uchino M, Christen WG, Schaumberg DA. Long-term Natural History of Dry Eye Disease from the Patient's Perspective. Ophthalmology 2016;123(2):425-33.Abstract

PURPOSE: To describe the natural history of dry eye disease (DED), which chronically affects millions of people in the United States. DESIGN: This study is based on the Women's Health Study and Physicians' Health Studies, and uses questionnaires and medical records. PARTICIPANTS: A total of 398 men and 386 women who reported a diagnosis of DED and responded to a questionnaire about change in disease since diagnosis. METHODS: Three subscales were developed using factor analysis of questionnaire responses: ocular surface symptoms, vision-related symptoms, and social impact. We examined correlates of worsening on each subscale, obtained medical records from a subset of 261 study participants, and examined changes in clinical signs of DED over time. MAIN OUTCOME MEASURES: Worsening in ocular surface symptoms, vision-related symptoms, and social impact plus clinical signs. RESULTS: The average duration of DED of 10.5 years (standard deviation, 9.5 years). Worsening was reported by 24% for ocular surface symptoms, 29% for vision-related symptoms, and 10% for social impact. Factors associated with worsening on at least 2 of 3 subscales included a previous report of severe DED symptoms (odds ratio [OR], 2.17 for ocular surface symptoms; OR, 2.35 for vision-related symptoms), spending >$20 per month on DED treatments (OR, 1.80 for ocular surface symptoms; OR, 1.99 for vision-related symptoms), history of blepharitis or meibomian gland dysfunction (MGD) (OR, 1.57 for vision-related symptoms; OR, 2.12 for social impact), and use of systemic beta-blockers (OR, 1.62 for ocular surface symptoms; OR, 1.84 for vision-related symptoms; OR, 1.86 for the social impact of DED). Presence of corneal staining based on review of medical records was associated with use of level 2 or higher DED treatments (OR, 1.54; confidence interval [CI], 1.01-2.36), a previous report of severe DED symptoms (OR, 1.79; CI, 1.07-3.00), having a tear break-up test performed (OR, 2.73; CI, 1.72-4.36), and having blepharitis or MGD (OR, 0.59; CI, 0.35-0.98). CONCLUSIONS: A proportion of patients with DED experience worsening over time, tending to report with more severe symptoms earlier in the disease. Forthcoming data on the natural history of DED from prospective studies should help clarify some of the limitations of this retrospective study.

Lin YB, Gardiner MF. Fingernail-induced corneal abrasions: case series from an ophthalmology emergency department. Cornea 2014;33(7):691-5.Abstract
PURPOSE: Fingernail-induced corneal abrasions are one of the most common eye injuries that present to the emergency department, and yet there is little literature available to offer guidelines for management. We analyzed the treatment used in cases of fingernail-induced corneal abrasions that presented to the Massachusetts Eye and Ear Infirmary Emergency Department and studied its relationship to the development of complications such as recurrent erosion syndrome and infection. METHODS: We performed a retrospective review of 99 patients who presented to the Massachusetts Eye and Ear Infirmary Emergency Department with fingernail-induced corneal abrasions between January 1, 2009 and December 31, 2009. We followed the patients for 12 months and documented demographics, nature of the injury, treatment, and complications. RESULTS: The average age was 29.4 (range, 2-89) years. Forty-four percent (n = 44) were female and 56% (n = 55) were male. Of the 99 subjects, 39 had a full 12 month follow-up, and 7 developed a complication from the injury. Compared with the 32 subjects without complications, there was no difference in age or gender. However, there was a significant difference in that adults scratched by another adult were more highly represented in the group with complications (43%, n = 3/7 vs. 3%, n = 1/32; P = 0.0017). There was no significant difference in outcome by treatment used. CONCLUSIONS: This is the largest fingernail-induced corneal abrasion study completed to date. Patients are at risk of developing complications, but there is scant evidenced-based literature available for treating this common injury. Prospective trials should be performed to better optimize and standardize treatments.
Lin SR, Aldave AJ, Chodosh J. Recurrent corneal erosion syndrome. Br J Ophthalmol 2019;103(9):1204-1208.Abstract
Recurrent corneal erosion syndrome (RCES) is a disorder characterised by a dysfunctional epithelial ecosystem. It often begins after trauma, or in the setting of epithelial basement membrane degeneration or dystrophy. Historically, RCES has been understood as a structural derangement of the anterior corneal architecture. More recently, studies have demonstrated the important role of neuropeptides in corneal homoeostasis. Thus, RCES may also be understood as a disorder of corneal epithelial cell biology. Management of RCES can be challenging, but newer therapies have demonstrated improved efficacy for this condition. This review examines the aetiology and pathogenesis of RCES, and provides an update on current and emerging treatment modalities for the management of this disorder.
Lippestad M, Hodges RR, Utheim TP, Serhan CN, Dartt DA. Resolvin D1 Increases Mucin Secretion in Cultured Rat Conjunctival Goblet Cells via Multiple Signaling Pathways. Invest Ophthalmol Vis Sci 2017;58(11):4530-4544.Abstract
Purpose: Goblet cells in the conjunctiva secrete mucin into the tear film protecting the ocular surface. The proresolution mediator resolvin D1 (RvD1) regulates mucin secretion to maintain homeostasis during physiological conditions and in addition, actively terminates inflammation. We determined the signaling mechanisms used by RvD1 in cultured rat conjunctival goblet cells to increase intracellular [Ca2+] ([Ca2+]i) and induce glycoconjugate secretion. Methods: Increase in [Ca2+]i were measured using fura 2/AM and glycoconjugate secretion determined using an enzyme-linked lectin assay with the lectin Ulex Europaeus Agglutinin 1. Signaling pathways activated by RvD1 were studied after goblet cells were pretreated with signaling pathway inhibitors before stimulation with RvD1. The results were compared with results when goblet cells were stimulated with RvD1 alone and percent inhibition calculated. Results: The increase in [Ca2+]i stimulated by RvD1 was blocked by inhibitors to phospholipases (PL-) -D, -C, -A2, protein kinase C (PKC), extracellular signal-regulated kinases (ERK)1/2 and Ca2+/calmodulin-dependent kinase (Ca2+/CamK). Glycoconjugate secretion was significantly inhibited by PLD, -C, -A2, ERK1/2 and Ca2+/CamK, but not PKC. Conclusions: We conclude that RvD1 increases glycoconjugate secretion from goblet cells via multiple signaling pathways including PLC, PLD, and PLA2, as well as their signaling components ERK1/2 and Ca2+/CamK to preserve the mucous layer and maintain homeostasis by protecting the eye from desiccating stress, allergens, and pathogens.
Lippestad M, Hodges RR, Utheim TP, Serhan CN, Dartt DA. Signaling pathways activated by resolvin E1 to stimulate mucin secretion and increase intracellular Ca in cultured rat conjunctival goblet cells. Exp Eye Res 2018;173:64-72.Abstract
Glycoconjugate mucin secretion from conjunctival goblet cells is tightly regulated by nerves and specialized pro-resolving mediators (SPMs) to maintain ocular surface health. Here we investigated the actions of the SPM resolvin E1 (RvE1) on cultured rat conjunctival goblet cell glycoconjugate secretion and intracellular [Ca] ([Ca]) and the signaling pathways used by RvE1. Goblet cells were cultured from rat conjunctiva in RPMI medium. The amount of RvE1-stimulated glycoconjugate mucin secretion was determined using an enzyme-linked lectin assay with Ulex Europaeus Agglutinin 1 lectin. Cultured goblet cells were also incubated with the Ca indicator dye fura 2/AM and [Ca] was measured. Cultured goblet cells were incubated with inhibitors to phospholipase (PL-) C, D, and A2 signaling pathways. RvE1 stimulated glycoconjugate secretion in a concentration dependent manner and was inhibited with the Ca chelator BAPTA. The Ca response was also increased in a concentration manner when stimulated by RvE1. Inhibition of PLC, PLD, and PLA2, but not Ca/calmodulin-dependent kinase blocked RvE1-stimulated increase in [Ca] and glycoconjugate secretion. We conclude that under normal, physiological conditions RvE1 stimulates multiple pathways to increase glycoconjugate secretion and [Ca]. RvE1 could be an important regulator of goblet cell glycoconjugate mucin secretion to maintain ocular surface health.
Liu Y, Kam WR, Fernandes P, Sullivan DA. The Effect of Solithromycin, a Cationic Amphiphilic Drug, on the Proliferation and Differentiation of Human Meibomian Gland Epithelial Cells. Curr Eye Res 2018;43(6):683-688.Abstract
PURPOSE: We previously discovered that azithromycin (AZM) acts directly on immortalized human meibomian gland epithelial cells (IHMGECs) to stimulate their lipid and lysosome accumulation and overall differentiation. We hypothesize that this phospholipidosis-like effect is due to AZM's cationic amphiphilic drug (CAD) nature. If our hypothesis is correct, then other CADs (e.g., solithromycin [SOL]) should be able to duplicate AZM's action on IHMGECs. Our purpose was to test this hypothesis. MATERIALS AND METHODS: IHMGECs were cultured in the presence of vehicle or SOL (2, 10, or 20 µg/ml) for up to 7 days under proliferating or differentiating conditions. Positive (epidermal growth factor and bovine pituitary extract for proliferation; AZM for differentiation) and negative (vehicle) controls were included with the experiments. IHMGECs were evaluated for cell number, neutral lipid content, and lysosome accumulation. RESULTS: Our results demonstrate that SOL induces a rapid and dose-dependent increase in the accumulation of neutral lipids and lysosomes in HMGECs. The lysosomal effects were most prominent with the 10 and 20 µg/ml doses, and occurred earlier (i.e., 1 day) with SOL than with the AZM (10 µg/ml) control. The effects of SOL and AZM on IHMGEC differentiation were essentially the same after 3 days of culture. SOL did not influence the proliferation of HMGECs during a 7-day time period. CONCLUSIONS: Our results support our hypothesis that SOL, a CAD, is able to reproduce AZM's impact on lysosome and lipid accumulation, as well as the differentiation, of HMGECs. The effect of SOL on lysosome appearance was faster than that of AZM.
Liu Y, Kam WR, Ding J, Sullivan DA. Can tetracycline antibiotics duplicate the ability of azithromycin to stimulate human meibomian gland epithelial cell differentiation?. Cornea 2015;34(3):342-6.Abstract

PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Liu S, Veldman P. Evidence-Based Endothelial Rehabilitation. Semin Ophthalmol 2017;32(1):96-103.Abstract

Endothelial keratoplasty (EK) has replaced penetrating keratoplasty (PKP) as the preferred surgical therapy for corneal endothelial dysfunction. However, recent nationwide corneal graft registry data showed few advantages to EK relative to PKP with respect to graft survival and visual outcomes. This article compares the published outcomes and complications of EK to those of PKP. EK demonstrates superior spectacle corrected visual outcomes, fast recovery, less graft rejection, and higher patient satisfaction, particularly in studies performed by high-volume surgeons/centers. Endothelial cell loss in EK, while higher at early time points, was equivalent or superior at five-years' follow-up and graft survival was equivalent to or superior to PKP in these centers/studies. Continued standardization and simplification of EK procedures may allow surgeons who perform a lower volume of EK to achieve results that mirror those of high-volume centers/surgeons and close the potential gap in outcomes demonstrated in the registry data.

Liu C, Chen Y, Kochevar IE, Jurkunas UV. Decreased DJ-1 leads to impaired Nrf2-regulated antioxidant defense and increased UV-A-induced apoptosis in corneal endothelial cells. Invest Ophthalmol Vis Sci 2014;55(9):5551-60.Abstract
PURPOSE: To investigate the role of DJ-1 in Nrf2-regulated antioxidant defense in corneal endothelial cells (CECs) at baseline and in response to ultraviolet A (UV-A)-induced oxidative stress. METHODS: DJ-1-deficient CECs were obtained by transfection of an immortalized normal human corneal endothelial cell line (HCECi) with DJ-1 small interfering RNA (siRNA) or by isolation of CECs from ex vivo corneas of DJ-1 knockout mice. Levels of reactive oxygen species (ROS), protein carbonyls, Nrf2 subcellular localization, Nrf2 target genes, and protein interaction between Keap1/Nrf2 and Cul3/Nrf2 were compared between normal and DJ-1-deficient CECs. Oxidative stress was induced by irradiating HCECi cells with UV-A, and cell death and levels of activated caspase3 and phospho-p53 were determined. RESULTS: DJ-1 siRNA-treated cells exhibited increased levels of ROS production and protein carbonyls as well as a 2.2-fold decrease in nuclear Nrf2 protein when compared to controls. DJ-1 downregulation led to attenuated gene expression of Nrf2 and its target genes HO-1 and NQO1. Similar levels of Nrf2 inhibitor, Keap1, and Cul3/Nrf2 and Keap1/Nrf2 were observed in DJ-1 siRNA-treated cells as compared to controls. Ultraviolet A irradiation resulted in a 3.0-fold increase in cell death and elevated levels of activated caspase3 and phospho-p53 in DJ-1 siRNA-treated cells compared to controls. CONCLUSIONS: Downregulation of DJ-1 impairs nuclear translocation of Nrf2, causing decreased antioxidant gene expression and increased oxidative damage. The decline in DJ-1 levels leads to heightened CEC susceptibility to UV-A light by activating p53-dependent apoptosis. Targeting the DJ-1-Nrf2 axis may provide a potential therapeutic approach for enhancing antioxidant defense in corneal endothelial disorders.
Liu C, Vojnovic D, Kochevar IE, Jurkunas UV. UV-A Irradiation Activates Nrf2-Regulated Antioxidant Defense and Induces p53/Caspase3-Dependent Apoptosis in Corneal Endothelial Cells. Invest Ophthalmol Vis Sci 2016;57(4):2319-27.Abstract

PURPOSE: To examine whether Nrf2-regulated antioxidant defense and p53 are activated in human corneal endothelial cells (CEnCs) by environmental levels of ultraviolet A (UV-A), a known stimulator of oxidative stress. METHODS: Immortalized human CEnCs (HCEnCi) were exposed to UV-A fluences of 2.5, 5, 10, or 25 J/cm2, then allowed to recover for 3 to 24 hours. Control HCEnCi did not receive UV-A. Reactive oxygen species (ROS) were measured using H2DCFDA. Cell cytotoxicity was evaluated by lactate dehydrogenase (LDH) release. Levels of Nrf2, HO-1, NQO-1, p53, and caspase3 were detected by immunnoblotting or real-time PCR. Activated caspase3 was measured by immunoblotting and a fluorescence assay. RESULTS: Exposure of HCEnCi to 5, 10, and 25 J/cm2 UV-A increased ROS levels compared with controls. Nrf2, HO-1, and NQO-1 mRNA increased 1.7- to 3.2-fold at 3 and 6 hours after irradiation with 2.5 and 5 J/cm2 UV-A. At 6 hours post irradiation, UV-A (5 J/cm2) enhanced nuclear Nrf2 translocation. At 24 hours post treatment, UV-A (5, 10, and 25 J/cm2) produced a 1.8- to 2.8-fold increase in phospho-p53 and a 2.6- to 6.0-fold increase in activated caspase3 compared with controls, resulting in 20% to 42% cell death. CONCLUSIONS: Lower fluences of UV-A induce Nrf2-regulated antioxidant defense and higher fluences activate p53 and caspase3, indicating that even near-environmental levels of UV-A may affect normal CEnCs. This data suggest that UV-A may especially damage cells deficient in antioxidant defense, and thus may be involved in the etiology of Fuchs' endothelial corneal dystrophy (FECD).

Liu Y, Chen D, Chen X, Kam WR, Hatton MP, Sullivan DA. Hypoxia: A breath of fresh air for the meibomian gland. Ocul Surf 2018;Abstract
PURPOSE: Optimal meibomian gland (MG) function is critically important for the health and wellbeing of the ocular surface. We hypothesize that low oxygen (O) conditions promote the function of human MG epithelial cells (HMGECs) and that human MGs exist in a relatively hypoxic environment. The purpose of this study was to test our hypotheses. METHODS: We used human and mouse eyelid segments, and immortalized human MG epithelial cells (IHMGECs) in our studies. To evaluate oxygen (O) levels in the mouse MG and vicinity, we injected pimonidazole (pimo), a hypoxia marker, before sacrifice. Human eyelid samples were stained with the hypoxia marker glucose transporter 1 (Glut-1). To determine the effect of low O levels on IHMGECs, we cultured cells under proliferating and differentiating conditions in both normoxic (21% O) and hypoxic (3% O) conditions for 5-15 days. IHMGECs were evaluated for cell number, neutral lipid content, lysosome accumulation, expression of biomarker proteins and DNase II activity. RESULTS: Our results demonstrate that human and mouse MGs, but not the surrounding connective tissue, exist in a relatively hypoxic environment in vivo. In addition, our findings show that hypoxia does not influence IHMGEC numbers in basal or proliferating culture conditions, but does stimulate the expression of SREBP-1 in differentiating IHMGECs. Hypoxia also significantly increased DNase II activity, and apparently IHMGEC terminal differentiation. CONCLUSIONS: Our Results support our hypotheses, and indicate that relative hypoxia promotes MG function.
Liu C, Miyajima T, Melangath G, Miyai T, Vasanth S, Deshpande N, Kumar V, Ong Tone S, Gupta R, Zhu S, Vojnovic D, Chen Y, Rogan EG, Mondal B, Zahid M, Jurkunas UV. Ultraviolet A light induces DNA damage and estrogen-DNA adducts in Fuchs endothelial corneal dystrophy causing females to be more affected. Proc Natl Acad Sci U S A 2020;117(1):573-583.Abstract
Fuchs endothelial corneal dystrophy (FECD) is a leading cause of corneal endothelial (CE) degeneration resulting in impaired visual acuity. It is a genetically complex and age-related disorder, with higher incidence in females. In this study, we established a nongenetic FECD animal model based on the physiologic outcome of CE susceptibility to oxidative stress by demonstrating that corneal exposure to ultraviolet A (UVA) recapitulates the morphological and molecular changes of FECD. Targeted irradiation of mouse corneas with UVA induced reactive oxygen species (ROS) production in the aqueous humor, and caused greater CE cell loss, including loss of ZO-1 junctional contacts and corneal edema, in female than male mice, characteristic of late-onset FECD. UVA irradiation caused greater mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) damage in female mice, indicative of the sex-driven differential response of the CE to UVA, thus accounting for more severe phenotype in females. The sex-dependent effect of UVA was driven by the activation of estrogen-metabolizing enzyme CYP1B1 and formation of reactive estrogen metabolites and estrogen-DNA adducts in female but not male mice. Supplementation of -acetylcysteine (NAC), a scavenger of reactive oxygen species (ROS), diminished the morphological and molecular changes induced by UVA in vivo. This study investigates the molecular mechanisms of environmental factors in FECD pathogenesis and demonstrates a strong link between UVA-induced estrogen metabolism and increased susceptibility of females for FECD development.