Paraskevi E. Kolovou

ABCB5 is a limbal stem cell gene required for corneal development and repair

Corneal epithelial homeostasis and regeneration are sustained by limbal stem cells (LSCs), and LSC deficiency is a major cause of blindness worldwide. Transplantation is often the only therapeutic option available to patients with LSC deficiency. However, while transplant success depends foremost on LSC frequency within grafts, a gene allowing for prospective LSC enrichment has not been identified so far. Here we show that ATP-binding cassette, sub-family B, member 5 (ABCB5) marks LSCs and is required for LSC maintenance, corneal development and repair. Furthermore, we demonstrate that prospectively isolated human or murine ABCB5-positive LSCs possess the exclusive capacity to fully restore the cornea upon grafting to LSC-deficient mice in xenogeneic or syngeneic transplantation models. ABCB5 is preferentially expressed on label-retaining LSCs in mice and p63α-positive LSCs in humans. Consistent with these findings, ABCB5-positive LSC frequency is reduced in LSC-deficient patients. Abcb5 loss of function in Abcb5 knockout mice causes depletion of quiescent LSCs due to enhanced proliferation and apoptosis, and results in defective corneal differentiation and wound healing. Our results from gene knockout studies, LSC tracing and transplantation models, as well as phenotypic and functional analyses of human biopsy specimens, provide converging lines of evidence that ABCB5 identifies mammalian LSCs. Identification and prospective isolation of molecularly defined LSCs with essential functions in corneal development and repair has important implications for the treatment of corneal disease, particularly corneal blindness due to LSC deficiency.

Opposing Roles for Membrane Bound and Soluble Fas Ligand in Glaucoma-Associated Retinal Ganglion Cell Death

Glaucoma, the most frequent optic neuropathy, is a leading cause of blindness worldwide. Death of retinal ganglion cells (RGCs) occurs in all forms of glaucoma and accounts for the loss of vision, however the molecular mechanisms that cause RGC loss remain unclear. The pro-apoptotic molecule, Fas ligand, is a transmembrane protein that can be cleaved from the cell surface by metalloproteinases to release a soluble protein with antagonistic activity. Previous studies documented that constitutive ocular expression of FasL maintained immune privilege and prevented neoangeogenesis. We now show that FasL also plays a major role in retinal neurotoxicity. Importantly, in both TNFα triggered RGC death and a spontaneous model of glaucoma, gene-targeted mice that express only full-length FasL exhibit accelerated RGC death. By contrast, FasL-deficiency, or administration of soluble FasL, protected RGCs from cell death. These data identify membrane-bound FasL as a critical effector molecule and potential therapeutic target in glaucoma.

Retinoblastoma cells are inhibited by aminoimidazole carboxamide ribonucleotide (AICAR) partially through activation of AMP-dependent kinase

5‐Aminoimidazole‐4‐carboxamide‐1‐β‐4‐ribofuranoside (AICAR), an analog of AMP, is widely used as an activator of AMP‐kinase (AMPK), a protein that regulates the responses of the cell to energy change. We studied the effects of AICAR on the growth of retinoblastoma cell lines (Y79, WERI, and RB143). AICAR inhibited Rb cell growth, induced apoptosis and S‐phase cell cycle arrest, and led to activation of AMPK. These effects were abolished by treatment with dypiridamole, an inhibitor that blocks entrance of AICAR into cells. Treatment with the adenosine kinase inhibitor 5‐iodotubericidin to inhibit the conversion of AICAR to ZMP (the direct activator of AMPK) reversed most of the growth‐inhibiting effects of AICAR, indicating that some of the antiproliferative effects of AICAR are mediated through AMPK activation. In addition, AICAR treatment was associated with inhibition of the mammalian target of rapamycin pathway, decreased phosphorylation of ribosomal protein‐S6 and 4E‐BP1, down‐regulation of cyclins A and E, and decreased expression of p21. Our results indicate that AICAR‐induced activation of AMPK inhibits retinoblastoma cell growth. This is one of the first descriptions of a nonchemotherapeutic drug with low toxicity that may be effective in treating Rb patients.—Theodoropoulou, S., Kolovou, P. E., Morizane, Y., Kayama, M., Nicolaou, F., Miller, J. W., Gragoudas, E., Ksander, B. R., Vavvas, D. G. Retinoblastoma cells are inhibited by aminoimidazole carboxamide ribonucleotide (AICAR) partially through activation of AMP‐dependent kinase. FASEB J. 24, 2620–2630 (2010). www.fasebj.org

Keratoprosthesis: A Review of Recent Advances in the Field

Since its discovery in the years of the French Revolution, the field of keratoprostheses has evolved significantly. However, the path towards its present state has not always been an easy one. Initially discarded for its devastating complications, the introduction of new materials and the discovery of antibiotics in the last century gave new life to the field. Since then, the use of keratoprostheses for severe ocular surface disorders and corneal opacities has increased significantly, to the point that it has become a standard procedure for corneal specialists worldwide. Although the rate of complications has significantly been reduced, these can impede the long-term success, since some of them can be visually devastating. In an attempt to overcome these complications, researchers in the field have been recently working on improving the design of the currently available devices, by introducing the use of new materials that are more biocompatible with the eye. Here we present an update on the most recent research in the field.

Corneal Anesthesia With Site 1 Sodium Channel Blockers and Dexmedetomidine

PURPOSE Amino-amide or amino-ester local anesthetics, which are currently used for topical ocular anesthesia, are short acting and may delay corneal healing with long-term use. In contrast, site 1 sodium channel blockers (S1SCBs) are potent local anesthetics with minimal adverse tissue reaction. In this study, we examined topical local anesthesia with two S1SCBs, tetrodotoxin (TTX) or saxitoxin (STX) individually or in combination with α2-adrenergic receptor agonists (dexmedetomidine or clonidine), and compared them with the amino-ester ocular anesthetic proparacaine. The effect of test solutions on corneal healing was also studied. METHODS Solutions of TTX ± dexmedetomidine, TTX ± clonidine, STX ± dexmedetomidine, dexmedetomidine, or proparacaine were applied to the rat cornea. Tactile sensitivity was measured by recording the blink response to probing of the cornea with a Cochet-Bonnet esthesiometer. The duration of corneal anesthesia was calculated. Cytotoxicity from anesthetic solutions was measured in vitro. The effect on corneal healing was measured in vivo after corneal debridement followed by repeated drug administration. RESULTS Addition of dexmedetomidine to TTX or STX significantly prolonged corneal anesthesia beyond that of either drug alone, whereas clonidine did not. Tetrodotoxin or STX coadministered with dexmedetomidine resulted in two to three times longer corneal anesthesia than did proparacaine. S1SCB-dexmedetomidine formulations were not cytotoxic. Corneal healing was not delayed significantly by any of the test solutions. CONCLUSIONS Coadministration of S1SCBs with dexmedetomidine provided prolonged corneal anesthesia without delaying corneal wound healing. Such formulations may be useful for the management of acute surgical and nonsurgical corneal pain.

A Murine Model for Metastatic Conjunctival Melanoma.

PURPOSE Conjunctival melanoma (CM) is an ocular malignancy with a high rate of local recurrences after treatment, and can give rise to deadly metastases. The establishment of a murine model will further our understanding of this disease and allow in vivo testing of new therapies. We therefore analyzed the ability of three CM cell lines to grow orthotopically and spread to distant sites. Furthermore, we determined the characteristics of the xenografts and their metastases. METHODS Orthotopic xenografts of human CM were established by subconjunctival injection of three different CM cell lines into NOD/SCID IL2 rγnull mice. Single-cell suspensions were generated from the primary tumors and placed subconjunctivally in another set of mice, which were then screened for metastases. The presence of melanoma markers was determined on the cell lines and during tumor development. RESULTS Subconjunctival injection of cultured CM cells into immunodeficient mice led to excellent subconjunctival tumor growth in all inoculated mice (n = 101) within 2 weeks; however, no metastases were found at the time of autopsy. Serial in vivo passage of primary tumor cells resulted in metastatic tumors in the draining lymph nodes (n = 21). The CM cell lines, as well as the tumor xenografts and their metastases, were positive for the melanoma markers HMB-45, S100B, and MART-1. Two cell lines and their corresponding xenografts carried a BRAF mutation, the third showed an NRAS mutation. CONCLUSIONS We established a murine model for CM that shows excellent formation of metastases in a pattern that accurately resembles metastatic human CM following in vivo passaging.

Boston Keratoprosthesis Type 1 in Chemical Burns.

Purpose: To describe and further analyze the long-term results in visual acuity (VA), anatomical retention, and rate of complications from patients who underwent Boston keratoprosthesis (B-Kpro) type 1 after ocular chemical burns in the Dominican Republic. Methods: A retrospective review of 42 eyes (22 OD:20 OS) of 36 patients who underwent B-Kpro type 1 implantation after severe ocular burn at Hospital Elías Santana in Santo Domingo, Dominican Republic, between April 2006 and October 2014, were included. Results: Demographics, VA, anatomical retention, and the rates of postoperative complications and concurrent surgeries were evaluated. Conclusions: The excellent anatomical retention rates and visual outcomes presented in this study support the remarkable capability of B-Kpro type 1 to restore functional VA in eyes with severe chemical injuries. However, strict control of the postoperative complications is necessary for long-term success. In conclusion, the use of a B-Kpro type 1 after severe chemical burn is a viable option in patients otherwise condemned to the high risk of failure associated with conventional corneal grafts.

Expression of Multidrug Resistance Transporter ABCB5 in a Murine Model of Human Conjunctival Melanoma

Conjunctival melanoma (CM) is a rare ocular malignancy with a high tendency to reoccur locally and with a high risk of metastatic disease. Metastases are often unresponsive to conventional treatment. Recently, an animal model was set up using human CM cells. Orthotopic xenografts from human CM were created by subconjunctival injection of three different CM cell lines into NOD.Cg-Prkdcscid IL2rgtm1Wjl/SzJ (NSG) mice. Subconjunctival injection of cultured CM cells led to excellent subconjunctival growth, but no metastases were found. When single-cell suspensions were obtained from the subconjunctival xenografts and passaged in vivo, all mice developed metastases. As recent findings indicate that cancer stem cells are linked to tumor recurrences, we used this new murine model to determine the expression of the stem cell marker ABCB5 during tumor progression. Expression of the ABCB5 protein was determined in three cell lines and during different stages of tumor development as observed in our model. All three cell lines contained a subpopulation of cells positive for ABCB5. During tumor development, expression of ABCB5 increased during phases of tumor expansion. Furthermore, expression of ABCB5 was increased in metastases. Using this model for CM, we were able to initiate metastatic spread and determine the expression of the stem cell marker ABCB5 during different stages of tumor development, identifying ABCB5 as a potential novel therapeutic target. This study illustrates the potential of our newly established murine model.

Abstract 4363: Identification of ABCB5 multidrug transporter in retinoblastoma

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Purpose: Retinoblastoma (Rb) is the most common intraocular tumor in children. Chemotherapy has improved the outcome for both unilateral and bilateral early stage disease, yet late stage bilateral Rb remains difficult to treat, and metastasis are often fatal. Treatment with carboplatin, vincristine and etoposide is only effective in 40% of bilateral Rb patients and results in a high incidence of secondary malignancies. ATP-binding cassette (ABC) transporters act as efflux pumps of different substrates, including drugs. ABCB5, a member of the ABC-B subfamily, is expressed on both normal tissues (CNS, mammary gland, testis, retina) and in skin melanoma, where it identifies a subpopulation of cancer stem cells with enhanced tumorigenicity. Unexpectedly, the retina displays the highest level of ABCG5 in normal tissues, suggesting it might be expressed in Rb tumors. Here we identify for the first time in Rb, an ABCB5+ subpopulation of cells that we predict will possess drug resistance and/or enhanced tumorigenicity. Methods: Rb143, Rb116, Rb125 and Rb107 cell lines were developed in our laboratory from primary explants recovered from patients with large tumors. Sequencing of the Rb gene (27 exons) was used to validate all Rb cell lines. Flow cytometry was used to identify ABCB5+ cells using a monoclonal anti-ABCB5 antibody or isotype control. Calcein AM and/or Aqua fluorescent dye was used to determine viability. ABCB5 pump function was determined by using Calcein AM, a pump substrate that becomes fluorescent within viable cells. Optical coherence tomography (OCT) was used to quantitate the size of intraocular tumors, following orthotopic injection into the sub-retinal space of NOD-scid IL2rg−/− mice. Results: All Rb cell lines possessed a subpopulation of ABCB5+ cells with a frequency ranging from 3-12%. To determine if the ABCB5 pump was functional, Rb cells were cultured for 30 mins with Calcein AM (ABCB5 pump substrate). ABCB5 positive, but not negative, Rb cells excluded the Calcein AM. The ABCB5 pump mediated Calcein exclusion, which was terminated by the addition of an ABCB5 specific blocking antibody. Rb143 cells (originally 3% ABCB5+ cells) were separated by cell sorting into (i) ABCB5+ cells (90% pure), and (ii) ABCB5- cells (100% pure). To test the growth potential and drug resistance of these two subpopulations in vivo, we developed a murine transient retinal detachment model. Injection of 50μl of PBS into the sub-retinal space induces a retinal detachment, but as the PBS is subsequently absorbed over the next 24 hrs the retina reattaches. Up to 1×106 cells were orthotopically injected beneath the detached retina, resulting in tumor growth in < 1 wk. This model will allow us to determine the drug resistance and growth potential of ABCB5+ and ABCB5- Rb cells. Conclusion: Retinoblastoma possesses a small sub-population of ABCB5+ tumor cells, which displays an active pump with the potential of drug resistance and preferential tumor growth. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4363.