Cornea Publications

Tahvildari M, Emami-Naeini P, Omoto M, Mashaghi A, Chauhan SK, Dana R. Treatment of donor corneal tissue with immunomodulatory cytokines: a novel strategy to promote graft survival in high-risk corneal transplantation. Sci Rep 2017;7(1):971.Abstract

Antigen-presenting cells (APCs) play an important role in transplant rejection and tolerance. In high-risk corneal transplantation, where the graft bed is inflamed and vascularized, immature APCs in the donor corneal stroma quickly mature and migrate to lymphoid tissues to sensitize host T cells. In this study, using a mouse model of corneal transplantation, we investigated whether enrichment of tolerogenic APCs (tolAPCs) in donor corneas can enhance graft survival in corneal allograft recipients with inflamed graft beds. Treatment of donor corneas with interleukin-10 (IL-10) and transforming growth factor-β1 (TGFβ1) altered the phenotype and function of tissue-residing APCs. Transplantation of these tolAPC-enriched corneas decreased frequencies of interferon gamma (IFNγ)(+) effector T cells (Teffs), as well as allosensitization in the hosts, diminished graft infiltration of CD45(+) and CD4(+) cells, and significantly improved corneal allograft survival compared to saline-injected controls. These data provide a novel approach for tolAPC-based immunotherapy in transplantation by direct cytokine conditioning of the donor tissue.

Rao R, Borkar DS, Colby KA, Veldman PB. Descemet Membrane Endothelial Keratoplasty After Failed Descemet Stripping Without Endothelial Keratoplasty. Cornea 2017;Abstract

PURPOSE: To describe the clinical course, surgical experience, and postoperative outcomes of 3 patients with Fuchs endothelial dystrophy who underwent Descemet membrane endothelial keratoplasty (DMEK) after failed Descemet stripping without endothelial keratoplasty. METHODS: Three patients who underwent DMEK for management of persistent corneal edema after deliberate Descemet stripping in the setting of Fuchs endothelial dystrophy were identified. Patients were examined at day 1, week 1, and months 1, 3, and 6 after DMEK. Visual acuity, central corneal thickness (CCT), and evaluation of central corneal endothelial cell counts were recorded. RESULTS: Two women and one man, aged 56, 72, and 68 years, were included. The time interval between primary Descemet stripping and DMEK ranged from 3.5 to 8 months. Preoperative visual acuities were 20/200, 20/300, and 20/80. Immediately before DMEK, no patients had countable central endothelial cells, and CCTs were 825, 1034, and 878 μm. After DMEK, all patients had improvement in visual acuity to 20/70, 20/20, and 20/20 with CCTs of 529, 504, and 528. The postoperative period in the first case was notable for the immediate development of a pigmented pupillary membrane with posterior synechiae, as well as cystoid macular edema, of uncertain chronicity, noted 1 month postoperatively. The second case also developed posterior synechiae. Two cases completed 6-month endothelial cell counts totaling 2200 and 3114 cells per square millimeter (endothelial cell loss of 13% and 5.3%). CONCLUSIONS: DMEK is a reliable procedure to facilitate corneal rehabilitation and visual recovery in the event of poor corneal clearance after Descemet stripping without endothelial keratoplasty.

Paschalis EI, Zhou C, Lei F, Scott N, Kapoulea V, Robert M-C, Vavvas D, Dana R, Chodosh J, Dohlman CH. Mechanisms of Retinal Damage after Ocular Alkali Burns. Am J Pathol 2017;Abstract

Alkali burns to the eye constitute a leading cause of worldwide blindness. In recent case series, corneal transplantation revealed unexpected damage to the retina and optic nerve in chemically burned eyes. We investigated the physical, biochemical, and immunological components of retinal injury after alkali burn and explored a novel neuroprotective regimen suitable for prompt administration in emergency departments. Thus, in vivo pH, oxygen, and oxidation reduction measurements were performed in the anterior and posterior segment of mouse and rabbit eyes using implantable microsensors. Tissue inflammation was assessed by immunohistochemistry and flow cytometry. The experiments confirmed that the retinal damage is not mediated by direct effect of the alkali, which is effectively buffered by the anterior segment. Rather, pH, oxygen, and oxidation reduction changes were restricted to the cornea and the anterior chamber, where they caused profound uveal inflammation and release of proinflammatory cytokines. The latter rapidly diffuse to the posterior segment, triggering retinal damage. Tumor necrosis factor-α was identified as a key proinflammatory mediator of retinal ganglion cell death. Blockade, by either monoclonal antibody or tumor necrosis factor receptor 1 and 2 gene knockout, reduced inflammation and retinal ganglion cell loss. Intraocular pressure elevation was not observed in experimental alkali burns. These findings illuminate the mechanism by which alkali burns cause retinal damage and may have importance in designing therapies for retinal protection.

Gouveia RM, González-Andrades E, Cardona JC, González-Gallardo C, Ionescu AM, Garzon I, Alaminos M, González-Andrades M, Connon CJ. Controlling the 3D architecture of Self-Lifting Auto-generated Tissue Equivalents (SLATEs) for optimized corneal graft composition and stability. Biomaterials 2017;121:205-219.Abstract

Ideally, biomaterials designed to play specific physical and physiological roles in vivo should comprise components and microarchitectures analogous to those of the native tissues they intend to replace. For that, implantable biomaterials need to be carefully designed to have the correct structural and compositional properties, which consequently impart their bio-function. In this study, we showed that the control of such properties can be defined from the bottom-up, using smart surface templates to modulate the structure, composition, and bio-mechanics of human transplantable tissues. Using multi-functional peptide amphiphile-coated surfaces with different anisotropies, we were able to control the phenotype of corneal stromal cells and instruct them to fabricate self-lifting tissues that closely emulated the native stromal lamellae of the human cornea. The type and arrangement of the extracellular matrix comprising these corneal stromal Self-Lifting Analogous Tissue Equivalents (SLATEs) were then evaluated in detail, and was shown to correlate with tissue function. Specifically, SLATEs comprising aligned collagen fibrils were shown to be significantly thicker, denser, and more resistant to proteolytic degradation compared to SLATEs formed with randomly-oriented constituents. In addition, SLATEs were highly transparent while providing increased absorption to near-UV radiation. Importantly, corneal stromal SLATEs were capable of constituting tissues with a higher-order complexity, either by creating thicker tissues through stacking or by serving as substrate to support a fully-differentiated, stratified corneal epithelium. SLATEs were also deemed safe as implants in a rabbit corneal model, being capable of integrating with the surrounding host tissue without provoking inflammation, neo-vascularization, or any other signs of rejection after a 9-months follow-up. This work thus paves the way for the de novo bio-fabrication of easy-retrievable, scaffold-free human tissues with controlled structural, compositional, and functional properties to replace corneal, as well as other, tissues.

Argüeso P. Proteolytic activity in the meibomian gland: Implications to health and disease. Exp Eye Res 2017;Abstract

The function of the meibomian gland in the upper and lower eyelids is critical to maintaining homeostasis at the ocular surface. Highly specialized meibocytes within the gland must differentiate and accumulate intracellular lipid droplets that are released into the tear film following rupture of the cell membrane. Proteases and their inhibitors have been recognized as key players in remodeling extracellular matrices and promoting the normal integrity of glandular tissue. They modulate a wide range of biological processes, such as cell proliferation and differentiation, and can contribute to disease when aberrantly expressed. Deciphering the role of proteolytic activity in the meibomian gland offers an opportunity to gain a more comprehensive and fundamental understanding of the developmental, physiological, and pathological processes associated with this gland.

Amouzegar A, Mittal SK, Sahu A, Sahu SK, Chauhan SK. Mesenchymal Stem Cells Modulate Differentiation of Myeloid Progenitor Cells during Inflammation. Stem Cells 2017;Abstract

Mesenchymal stem cells (MSCs) possess distinct immunomodulatory properties and have tremendous potential for use in therapeutic applications in various inflammatory diseases. MSCs have been shown to regulate pathogenic functions of mature myeloid inflammatory cells, such as macrophages and neutrophils. Intriguingly, the capacity of MSCs to modulate differentiation of myeloid progenitors to mature inflammatory cells remains unknown to date. Here, we report the novel finding that MSCs inhibit the expression of differentiation markers on myeloid progenitors under inflammatory conditions. We demonstrate that the inhibitory effect of MSCs is dependent on direct cell-cell contact and that this intercellular contact is mediated through interaction of CD200 expressed by MSCs and CD200R1 expressed by myeloid progenitors. Further, using an injury model of sterile inflammation, we show that MSCs promote myeloid progenitor frequencies and suppress infiltration of inflammatory cells in the inflamed tissue. We also find that downregulation of CD200 in MSCs correlates with abrogation of their immunoregulatory function. Collectively, our study provides unequivocal evidence that MSCs inhibit differentiation of myeloid progenitors in the inflammatory environment via CD200-CD200R1 interaction. This article is protected by copyright. All rights reserved.

Kim EC, Toyono T, Berlinicke CA, Zack DJ, Jurkunas U, Usui T, Jun AS. Screening and Characterization of Drugs That Protect Corneal Endothelial Cells Against Unfolded Protein Response and Oxidative Stress. Invest Ophthalmol Vis Sci 2017;58(2):892-900.Abstract

Purpose: To screen for and characterize compounds that protect corneal endothelial cells against unfolded protein response (UPR) and oxidative stress. Methods: Bovine corneal endothelial cells (BCECs) were treated for 48 hours with 640 compounds from a Food and Drug Administration (FDA)-approved drug library and then challenged with thapsigargin or H2O2 to induce UPR or oxidative stress, respectively. Cell viability was measured using the CellTiter-Glo survival assay. Selected "hits" were subjected to further dose-response testing, and their ability to modulate expression of UPR and oxidative stress markers was assessed by RT-PCR, Western blot, and measurement of protein carbonyl and 8-hydroxydeoxyguanosine (8-OHdG) adducts in immortalized human corneal endothelial cells (iHCECs). Results: Forty-one drugs at 20 μM and 55 drugs at 100 μM increased survival of H2O2-challenged cells, and 8 drugs at 20 μM and 2 drugs at 100 μM increased survival of thapsigargin-challenged cells, compared with untreated control cells. Nicergoline, ergothioneine, nimesulide, oxotremorine, and mefenamic acid increased survival of both H2O2- and thapsigargin-challenged cells. Oxotremorine altered DNA damage inducible 3 (CHOP) gene expression, glucose-regulated protein 78 kDa (GRP78) and activating transcription factor 4 (ATF4) protein expression, and protein carbonyl and 8-OHdG levels. Mefenamic acid altered GRP78 protein expression and protein carbonyl and 8-OHdG levels. Conclusions: Oxotremorine and mefenamic acid are potential survival factors for corneal endothelial cells under UPR and oxidative stress. The described assay can be further expanded to screen additional drugs for potential therapeutic effect in corneal endothelial diseases such as Fuchs' endothelial corneal dystrophy.

Chen Y, Chauhan SK, Tan X, Dana R. Interleukin-7 and -15 maintain pathogenic memory Th17 cells in autoimmunity. J Autoimmun 2017;77:96-103.Abstract

Th17 cells are principal mediators of many autoimmune conditions. Recently, memory Th17 cells have been revealed as crucial in mediating the chronicity of various refractory autoimmune disorders; however, the underlying mechanisms maintaining memory Th17 cells have remained elusive. Here, using a preclinical model of ocular autoimmune disease we show that both IL-7 and IL-15 are critical for maintaining pathogenic memory Th17 cells. Neutralization of these cytokines leads to substantial reduction of memory Th17 cells; both IL-7 and IL-15 provide survival signals via activating STAT5, and IL-15 provides additional proliferation signals via activating both STAT5 and Akt. Topical neutralization of ocular IL-7 or IL-15 effectively reduces memory Th17 cells at the inflammatory site and draining lymphoid tissues, while topical neutralization of IL-17 alone, the major pathogenic cytokine secreted by Th17 cells, does not diminish memory Th17 cells at the draining lymphoid tissues. Our results suggest that the effective removal of pathogenic memory Th17 cells via abolishing environmental IL-7 or IL-15 is likely to be a novel strategy in the treatment of autoimmune diseases.

Han K-Y, Tran JA, Chang J-H, Azar DT, Zieske JD. Potential role of corneal epithelial cell-derived exosomes in corneal wound healing and neovascularization. Sci Rep 2017;7:40548.Abstract

Specific factors from the corneal epithelium underlying the stimulation of stromal fibrosis and myofibroblast formation in corneal wound healing have not been fully elucidated. Given that exosomes are known to transfer bioactive molecules among cells and play crucial roles in wound healing, angiogenesis, and cancer, we hypothesized that corneal epithelial cell-derived exosomes may gain access to the underlying stromal fibroblasts upon disruption of the epithelial basement membrane and that they induce signaling events essential for corneal wound healing. In the present study, exosome-like vesicles were observed between corneal epithelial cells and the stroma during wound healing after corneal epithelial debridement. These vesicles were also found in the stroma following anterior stromal keratectomy, in which surgical removal of the epithelium, basement membrane, and anterior stroma was performed. Exosomes secreted by mouse corneal epithelial cells were found to fuse to keratocytes in vitro and to induce myofibroblast transformation. In addition, epithelial cell-derived exosomes induced endothelial cell proliferation and ex vivo aortic ring sprouting. Our results indicate that epithelial cell-derived exosomes mediate communication between corneal epithelial cells and corneal keratocytes as well as vascular endothelial cells. These findings demonstrate that epithelial-derived exosomes may be involved in corneal wound healing and neovascularization, and thus, may serve as targets for potential therapeutic interventions.

English JT, Norris PC, Hodges RR, Dartt DA, Serhan CN. Identification and Profiling of Specialized Pro-Resolving Mediators in Human Tears by Lipid Mediator Metabolomics. Prostaglandins Leukot Essent Fatty Acids 2017;117:17-27.Abstract

Specialized pro-resolving mediators (SPM), e.g. Resolvin D1, Protectin D1, Lipoxin A₄, and Resolvin E1 have each shown to be active in ocular models reducing inflammation. In general, SPMs have specific agonist functions that stimulate resolution of infection and inflammation in animal disease models. The presence and quantity of SPM in human emotional tears is of interest. Here, utilizing a targeted LC-MS-MS metabololipidomics based approach we document the identification of pro-inflammatory (Prostaglandins and Leukotriene B₄) and pro-resolving lipid mediators (D-series Resolvins, Protectin D1, and Lipoxin A₄) in human emotional tears from 12 healthy individuals. SPMs from the Maresin family (Maresin 1 and Maresin 2) were not present in these samples. Principal Component Analysis (PCA) revealed gender differences in the production of specific mediators within these tear samples as the SPMs were essentially absent in these female donors. These results indicate that specific SPM signatures are present in human emotional tears at concentrations known to be bioactive. Moreover, they will help to further appreciate the mechanisms of production and action of SPMs in the eye, as well as their physiologic roles in human ocular disease resolution.

Zhou C, Robert M-C, Kapoulea V, Lei F, Stagner AM, Jakobiec FA, Dohlman CH, Paschalis EI. Sustained Subconjunctival Delivery of Infliximab Protects the Cornea and Retina Following Alkali Burn to the Eye. Invest Ophthalmol Vis Sci 2017;58(1):96-105.Abstract

Purpose: Tumor necrosis factor (TNF)-α is upregulated in eyes following corneal alkali injury and contributes to corneal and also retinal damage. Prompt TNF-α inhibition by systemic infliximab ameliorates retinal damage and improves corneal wound healing. However, systemic administration of TNF-α inhibitors carries risk of significant complications, whereas topical eye-drop delivery is hindered by poor ocular bioavailability and the need for patient adherence. This study investigates the efficacy of subconjunctival delivery of TNF-α antibodies using a polymer-based drug delivery system (DDS). Methods: The drug delivery system was prepared using porous polydimethylsiloxane/polyvinyl alcohol composite fabrication and loaded with 85 μg of infliximab. Six Dutch-belted pigmented rabbits received ocular alkali burn with NaOH. Immediately after the burn, subconjunctival implantation of anti-TNF-α DDS was performed in three rabbits while another three received sham DDS (without antibody). Rabbits were followed with photography for 3 months. Results: After 3 months, the device was found to be well tolerated by the host and the eyes exhibited less corneal damage as compared to eyes implanted with a sham DDS without drug. The low dose treatment suppressed CD45 and TNF-α expression in the burned cornea and inhibited retinal ganglion cell apoptosis and optic nerve degeneration, as compared to the sham DDS treated eyes. Immunolocalization revealed drug penetration in the conjunctiva, cornea, iris, and choroid, with residual infliximab in the DDS 3 months after implantation. Conclusions: This reduced-risk biologic DDS improves corneal wound healing and provides retinal neuroprotection, and may be applicable not only to alkali burns but also to other inflammatory surgical procedures such as penetrating keratoplasty and keratoprosthesis implantation.

Lambiase A, Sullivan BD, Schmidt TA, Sullivan DA, Jay GD, Truitt ER, Bruscolini A, Sacchetti M, Mantelli F. A Two-Week, Randomized, Double-masked Study to Evaluate Safety and Efficacy of Lubricin (150 μg/mL) Eye Drops Versus Sodium Hyaluronate (HA) 0.18% Eye Drops (Vismed®) in Patients with Moderate Dry Eye Disease. Ocul Surf 2017;15(1):77-87.Abstract

PURPOSE: The objective of this clinical trial (NCT02507934) was to assess the efficacy and safety of recombinant human lubricin as compared to a 0.18% sodium hyaluronate (HA) eye drop in subjects with moderate dry eye disease (DED). METHODS: DEWS Grade 2-3 subjects were randomized to use lubricin (N=19, 51.9 ± 11.8 years) or HA (N=20, 61.8 ± 13.3 years). After a saline washout period, subjects administered BID therapy for 7 days, followed by instillation as needed (2-6 drops per eye) for 7 days. Visual analog scale (VAS) including foreign body sensation, burning/stinging, itching, pain, sticky feeling, blurred vision and photophobia were primary outcomes, with secondary endpoints of corneal fluorescein staining, Schirmer test, tear film breakup time (TFBUT), eyelid and conjunctival erythema and number of instillations compared at day 14. RESULTS: The primary endpoint was met. Lubricin supplementation achieved greater than a 72% reduction from baseline in foreign body sensation (P<.013), burning/stinging, pain, sticky feeling (P<.0432), blurred vision (P<.0013), and photophobia (P<.011) in at least one eye. Lubricin also showed significant improvement in fluorescein staining (OD/OS: 43.8%/50.0%, vs. 26.5%/23.3%, P<.0398, P<.0232), TFBUT (P<.010), SANDE frequency (P<.0435), eyelid erythema (P<.004), conjunctival erythema (P<.0013), and instillations (P<.04) as compared to HA. No treatment-related adverse events occurred during the investigation. CONCLUSIONS: Recombinant human lubricin was shown to produce significant improvement in both signs and symptoms of dry eye disease as compared to HA.

Ji YW, Mittal SK, Hwang HS, Chang E-J, Lee JH, Seo Y, Yeo A, Noh H, Lee HS, Chauhan SK, Lee HK. Lacrimal gland-derived IL-22 regulates IL-17-mediated ocular mucosal inflammation. Mucosal Immunol 2017;Abstract

Inflammatory damage of mucosal surface of the eye is a hallmark of dry eye disease (DED) and, in severe cases, can lead to significant discomfort, visual impairment, and blindness. DED is a multifactorial autoimmune disorder with a largely unknown pathogenesis. Using a cross-sectional patient study and a well-characterized murine model of DED, herein we investigated the immunoregulatory function of interleukin-22 (IL-22) in the pathogenesis of DED. We found that IL-22 levels were elevated in lacrimal fluids of DED patients and inversely correlated with severity of disease. Acinar cells of the lacrimal glands (LGs), not inflammatory immune cells, are the primary source of IL-22, which suppresses inflammation in ocular surface epithelial cells upon desiccating stress. Moreover, loss of function analyses using IL-22 knockout mice demonstrated that IL-22 is essential for suppression of ocular surface infiltration of Th17 cells and inhibition of DED induction. Our novel findings elucidate immunoregulatory function of LG-derived IL-22 in inhibiting IL-17-mediated ocular surface epitheliopathy in DED thus making IL-22 a new relevant therapeutic target.Mucosal Immunology advance online publication, 4 January 2017; doi:10.1038/mi.2016.119.

Inomata T, Mashaghi A, Di Zazzo A, Lee S-M, Chiang H, Dana R. Kinetics of Angiogenic Responses in Corneal Transplantation. Cornea 2017;36(4):491-496.Abstract

PURPOSE: To delineate and compare the kinetics of corneal angiogenesis after high-risk (HR) versus low-risk (LR) corneal transplantation. METHODS: In mice, intrastromal sutures were placed in the recipient graft bed 2 weeks before allogeneic transplantation to induce angiogenesis and amplify the risk of graft rejection. Control (LR) graft recipients did not undergo suture placement, and thus the host bed remained avascular at the time of transplantation. Graft hemangiogenesis and opacity scores were evaluated for 8 weeks by slit-lamp biomicroscopy. Immunohistochemistry was used to measure CD31 (blood vessels) and LYVE-1 (lymphatic vessels) cells. RESULTS: Biphasic kinetics were observed for hemangiogenesis in both HR and LR transplant recipients using clinical and immunohistochemical assessments. The biphasic kinetics were composed of a rise-fall (phase 1) followed by a second rise (phase 2) in the degree of vessels. Compared with LR recipients, HR recipients showed higher hemangiogenesis (whole cornea and graft) throughout 8 weeks. Analyzing grafts revealed sustained presence of lymphatic vessels in HR recipients; however, lymphatic neovessels regressed in LR recipients 2 weeks posttransplantation. In contrast to HR host beds, the LR host bed microenvironment cannot sustain the growth of lymphatic neovessels in allografts, whereas it can sustain continued hemangiogenesis. CONCLUSIONS: The sustained presence of lymphatic vessels in HR host beds can facilitate host immunity against allografts and is likely associated with ongoing higher risk of rejection of these grafts in the long term, suggesting that therapeutic interventions targeting inflammation and lymphatic vessels need to be sustained long term in the HR corneal transplant setting.