Session 412 Corneal Epithelium

Transcription

ARVO 2016 Annual Meeting Abstracts
412 Corneal Epithelium
Wednesday, May 04, 2016 8:30 AM–10:15 AM
Exhibit/Poster Hall Poster Session
Program #/Board # Range: 4342–4385/A0145–A0188
Organizing Section: Cornea
Program Number: 4342 Poster Board Number: A0145
Presentation Time: 8:30 AM–10:15 AM
Preservation of the stem cell population in tissue-engineered
human corneas
Gaëtan Le-Bel1, 2, Karine Zaniolo1, Francis Bisson2, Sylvain Guérin1,
Lucie Germain1, 2. 1Département d’Ophtalmologie, Université Laval,
CUO-Recherche/LOEX, Centre de recherche du CHU de QuébecUniversité Laval, Quebec, QC, Canada; 2Département de Chirurgie,
Université Laval, Centre de recherche en organogénèse expérimentale
de l’Université Laval/LOEX, Centre de recherche du CHU de
Québec-Université Laval, Quebec, QC, Canada.
Purpose: In order to maintain a good transparency, the stratified
epithelium of the tissue-engineered cornea yielded by the limbic
stem cells must renew itself on a regular basis. It has been shown that
monitoring the expression of ubiquitous transcription factors such
as Sp1 and NFI that play critical functions in cell proliferation and
migration, may be of a considerable interest in order to assess the
quality of the epithelial stem cells population that are to be selected
for the production of tissue engineered corneas. In the present study,
we compared the efficiency of different feeder layers (murine or
human) to maintain the proliferative properties of primary cultures of
human corneal epithelial stem cells in vitro.
Methods: A human corneal stem cells population (HCSCs; n = 1)
was grown as a monolayer in the presence of irradiated fibroblasts
of either mouse (i3T3) or human (irradiated Human Feeder Layer or
iHFL) origin. The cells were amplified through several passages until
they reached a high rate of replicative senescence. The morphological
and growth rate characteristics were evaluated at each cell passage.
Both the expression and DNA binding properties of Sp1 and NFI
were assessed using Western blot and electrophoretic mobility shift
assays, respectively. Gene expression of Sp1 and NFI was also
monitored by RT-qPCR.
Results: HCSCs grown in the presence of iHFL as a feeder layer
could be maintained until passage 8 before they reached terminal
differentiation whereas they stopped proliferating at passage 6 when
grown in the presence of i3T3. No difference could be observed in
the amount and DNA binding activity of Sp1 between the different
conditions selected. On the other hand, HCSCs have an increased
NFI binding activity and also express an additional band for NFI
in Western blot when they were grown in the presence of i3T3.
RT-qPCR analyses revealed a strong increase in the mRNA level of
the NFIX isoform when HCSCs are cultured with an i3T3 but not an
iHFL feeder layer.
Conclusions: The feeder layer made from irradiated human
fibroblasts appears to be the most effective for the maintenance of
the HCSCs proliferative capacity. This might be related to the fact
that expression of one of the NFI isoforms is considerably reduced in
cells grown in the presence of iHFL as this transcription factor is well
known to function as efficiently as a repressor than an activator of
gene transcription.
Commercial Relationships: Gaëtan Le-Bel, None; Karine Zaniolo;
Francis Bisson, None; Sylvain Guérin, None; Lucie Germain,
None
Avastin effects on human limbal epithelial cell function and
phenotype in vitro
Maria Notara, Gabriele Braun, Marie L. Dreisow, Felix Bock,
Claus Cursiefen. Ophthalmology, University Hospital of Cologne,
Cologne, Germany.
Purpose: Topical application of the VEGFA inhibitor bevacizumab
(Avastin) is used for antiangiogenic therapy at the cornea. While
clinical studies have suggested that this off-label use of Avastin is
well-tolerated with relatively few adverse effects, the impact of
the drug on limbal epithelial stem cells is unknown. In this study,
the effect of Avastin on the functionality and phenotype of limbal
epithelial cells was investigated
Methods: The effect of different concentrations of Avastin (0 mg/ml,
0.125mg/ml, 0.5mg/ml and 1mg/ml) on human limbal epithelial
cells was investigated in terms of proliferation using an alamar blue
assay and migration by a scratch wound assay (SWA). Changes in
phenotype were assessed using a colony forming efficiency (CFE)
assay as well as immunostaining and QPCR of markers associated
with basal limbal epithelial cells, differentiated corneal epithelial
cells and putative stem cells including integrinβ1, cytokeratin 3 and
ΔΝΡ63∝ respectively. The effect of the treatment in RNA expression
of VEGFA, C and D and their receptors VEGFR1, 2 and 3 was also
assessed.
Results: The different treatment groups featured no difference in
proliferation and CFE as well as morphology and marker expression
by immunostaining. A small but significant delay in wound healing
of all the Avastin treated groups was detected at an early timepoint
of 2h. QPCR assessment indicated a small but significant increase
of integrinβ1 and a strong expression in cytokeratin 3 with the
increasing Avastin concentrations while ΔΝΡ63∝ RNA levels
remained unaffected. VEGFA RNA expression also significantly
increased while VEGFC and D were unchanged. No differences were
observed in the expression of VEGFR1, 2 and 3.
Conclusions: This study exhibited previously unknown shortterm effects of Bevazizumab in cultured human limbal epithelial
cells: Avastin-induced a dose-responsive increase of cytokeratin
3 expression, indicating a shift towards a more differentiated
phenotype, as well as a delay in wound closure correlating with the
delayed wound healing observed clinically with the use of Avastin.
The increase in VEGFA RNA levels may reflect an autocrine
defence mechanism of the limbal epithelial cells as they attempt to
compensate for the neutralisation effect of Avastin. The data obtained
through this in vitro approach confirm the effects of Avastin
while elucidating phenotypical and functional changes in limbal
epithelial cells.
Commercial Relationships: Maria Notara; Gabriele Braun, None;
Marie L. Dreisow, None; Felix Bock, None; Claus Cursiefen, None
The role of the extracellular matrix on human corneal and
conjunctival epithelial plasticity
Tiago Ramos1, Stewart Rosalind1, Stephen Kaye1, 2, Sajjad Ahmad1, 2.
1
Eye and Vision Science, University of Liverpool, Liverpool, United
Kingdom; 2St Paul’s Eye Unit, Royal Liverpool University Hospital,
Liverpool, United Kingdom.
Purpose: To determine if epithelial cells (ECs) from the human
cornea and conjunctiva can be modulated in response to extracellular
matrix (ECM) cues in vitro. This was investigated in both established
human EC lines and primary ECs. These studies test the hypothesis
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that ocular surface ECs have a degree of plasticity. This is of
importance due to its clinical significance.
Methods: Human corneal and conjunctival EC lines (hTCE-Pi and
HCjE-Gi respectively) and primary corneal and conjunctival ECs
from fresh human cadaveric tissue (Liverpool Research Eye Bank)
were used for the purposes of these studies. All experiments were
performed in triplicate. ECs were seeded on plastic for 9 days.
Using an established protocol, cells were removed leaving the ECM
attached to the plastic. Fresh corneal ECs (CoEC) were then cultured
on ECM from conjunctival ECs (CjEC) for 5 days and vice versa.
Marker expression for corneal epithelium, keratin (K) 3 and K12;
conjunctival epithelium, K7 and K13, and MUC5AC) and stem cell
(SC) markers (p63 and ABCB5) was analysed using real-time PCR,
Western blots (WB) and flow cytometry (FC).
Results: The following statistically significant results (p value at
least <0.05; n=3) are from experiments performed for the human
corneal and conjunctival EC lines and these were verified on primary
ECs from fresh human cadaveric cornea and conjunctiva. CoECs
cultured on ECM from CjECs (compared to those cultured on CoEC
ECM) showed down-regulation of CoEC markers K3 (WB and FC)
and K12 (FC); up-regulation of CjEC markers K7 and K13 (PCR
and FC) and goblet cell marker MUC5AC (PCR); and up-regulation
of SC markers p63 (FC) and ABCB5 (PCR and FC). CjECs plated
on ECM from CoECs (compared to those cultured on CjEC ECM)
showed down-regulation of CjEC markers K7 and K13 (PCR, WB
and FC); down-regulation of CoEC markers K3 and K12 (WB and
FC); and up-regulation of SC markers p63 (WB and FC) and ABCB5
(PCR, WB and FC).
Conclusions: These results are consistent with our hypothesis
that ECM cues modulate the plasticity of ECs of the human ocular
surface. Moreover, there is some evidence from these results that
this plasticity involves a process of de-differentiation (increased
expression of SC markers). Further work is being conducted to
understand the mechanisms underlying this plasticity and the
composition of the ECM deposited by the cells that enables this.
Commercial Relationships: Tiago Ramos, None;
Stewart Rosalind, None; Stephen Kaye, None; Sajjad Ahmad,
None
Support: Crossley Barnes Foundation
Spheroidal cultivation of the human limbal epithelial niche from
small limbal tissue
Kazunari Higa1, 2, Hideyuki Miyashita2, Jun Shimazaki1,
Kazuo Tsubota2, Shigeto Shimmura2. 1Ophthalmology/Cornea Center,
Tokyo Dental College Ichikawa General Hospital, Ichikawa, Japan;
2
Ophthalmology, Keio University School of Medicine, Shinanomachi,
Japan.
Purpose: Stem cells have a specialized microenvironment for
maintaining self-renewal and multipotent capacities, which is called
as niche. The niche of corneal epithelial stem cell exists in the
limbus. We previously reported that the limbal phenotype including
stem/niche-like structure can be maintained in spheroids derived from
the human limbus, for up to 1 month in medium with KGF+Y27632
using matrigel (2014 ARVO). To improve the cultivation method,
we assumed a limited autologous tissue and performed spheroidal
cultivation from human small limbal tissue.
Methods: Spheroids were prepared from donor limbal tissue 2.5
millimeters in diameter used after cornea transplantation. After
the treatment with collagenase, epithelial cells and surrounding
cells were scraped from limbal tissue, and spread on matrigel.
Cells were cultivated using medium containing KGF and the Rho
kinase inhibitor Y27632. To detect the slow cycling cells, cultivated
spheroids were labeled by 5-bromo-2’-deoxyuridine (BrdU) in first
3 days. After 1 month, spheroids were observed by histochemical
analysis, cell cycle analysis, and colony forming efficiency.
Results: Uniformly small and round spheroids were formed even
from small limbal tissue. Efficiency of spheroidal formation was
differed in the derived site of the original limbus. Spheroids derived
from the high spheroidal formation site showed the large colony
forming efficiency and higher ratio of slow cycling cells. In addition,
N-cadherin was infrequently-expressed in epithelial spheroid.
Conclusions: The limbal niche can be maintained in spheroids for up
to 1 month from small limbal tissue, but spheroidal forming ability
was differed in different parts of the limbus.
Commercial Relationships: Kazunari Higa, None;
Hideyuki Miyashita, None; Jun Shimazaki, None; Kazuo Tsubota,
None; Shigeto Shimmura, None
Support: JSPS KAKENHI grant number:24592646
Effect of intact amniotic membrane orientation on the
phenotypic potential of cultured limbal cells
Zala Luznik1, Petra Schollmayer1, Marina Bertolin2, Elvira Maličev3,
Sofija Andjelić1, Andreja Nataša Kopitar4, Alojz Ihan4,
Stefano Ferrari2, Marko Hawlina1. 1Eye Hospital, University Medical
Centre, Ljubljana, Slovenia, Ljubljana, Slovenia; 2Fondazione Banca
degli Occhi del Veneto, Venice, Italy; 3Blood Transfusion Centre of
Slovenia, Ljubljana, Slovenia; 4Medical Faculty Ljubljana, Institute
of Microbiology and Immunology, Ljubljana, Slovenia.
Purpose: Amniotic membrane (AM) serves as a surrogate limbal
niche for successful limbal epithelium (LE) expansion. The AM has
an epithelial and a stromal side with different biological properties.
The effect of intact AM orientation on the phenotypic potential
of cultured limbal cells from limbal explants is still not well
determined. Thus, we hypothesized that a difference in expression
of epithelial, mesenchymal and putative stem cell markers would be
observed between cultures cultivated on intact AM (on both sides)
compared to cultures cultivated on plastic culture plates without
AM (control group).
Methods: The preserved cadaveric human corneoscleral rim of
9 donors was cut into 2 mm2 sized explant samples. Each limbal
explant was cultured on AM (N=11) or on culture plastic plates
(N=13) using only human serum supplemented culturing medium.
Intact AM was positioned with either AM intact epithelial side
up (N=5) or the AM stromal side up (N=6). The expression of
mesenchymal markers (CD73, CD90, CD105), proliferation and
putative progenitor markers (CD184, CD117), and epithelial
markers (MUC, CK7) was determined by flow cytometry.
Immunohistochemistry on secondary cultures on AM was carried out
with antibodies against pancytokeratin, p63, Ki67. Student’s t-test
was used for statistical analysis.
Results: On the 3rd culturing day cell outgrowth was observed in all
the limbal explants. After one-week cell proliferation accelerated and
a change in cell phenotype was observed. Cells cultivated on culture
plates without AM became more elongated and spindle shaped. The
cells cultivated on AM retained an epithelial cell structure, which
was further confirmed by histology examination. Expression of
mesenchymal markers was significantly higher (p < 0.05) in cultures
cultivated without AM. Histology revealed limbal epithelial growth
and p63, Ki67 positive cells on both sides of AM. However, no
statistically significant difference was observed between CD117 and
CD184 positive cells in all tested conditions.
Conclusions: Limbal cells cultivated on AM exhibited a lower
expression profile of mesenchymal cell markers than limbal cells
cultivated on plastic plates, which is consistent with our hypothesis.
Histology confirmed limbal epithelial cell growth on both sides of
AM, with no morphological differences, or positivity of cells for p63
and Ki67 being observed between the two tested sides.
Commercial Relationships: Zala Luznik, None;
Petra Schollmayer, None; Marina Bertolin, None; Elvira Maličev,
None; Sofija Andjelić, None; Andreja Nataša Kopitar, None;
Alojz Ihan, None; Stefano Ferrari, None; Marko Hawlina, None
Support: This research work is a result of doctoral research, in part
financed by the European Union, European Social Fund and the
Republic of Slovenia, Ministry for Education, Science and Sport in
the framework of the Operational programme for human resources
development for the period 2007 – 2013.
miR-31/FIH-1/P21 Axis Regulates the Self-Renewal and
Differentiation in the Human Llimbal Stem Cells
Zhiping Liu, Xiangyin Sha, Zhiqun Min, Huyong Zou, Ye Wen,
Jing Zeng. Ophthalmology, The second affiliated hospital of
Guangzhou Medical University, Guangzhou, China.
Purpose: In our previous studies, we have found that corneal
epithelial cells could be dedifferentiated and retained the
undifferentiated phenotype in ES microenvironment. Furthermore,
this culture system could maintain the stemness of human limbal
stem cells (LSCs) via telomerase/p21/mitochondria axis and the
activation of FAK/Wnt signaling pathways. Studies have shown
that microRNAs could regulate the functional properties of adult
stem cells. Based on our previous work, we propose to explore
the molecular mechanism of ES micro-environment regulates the
self-renewal and differentiation in LSCs.
Methods: The LSCs were cultured in different media, either CnT-20
medium or CnT-20 +20% ES culture supernatant (ESC-CM). Colony
formation assay was used to analyse cell proliferation. miR-31
mimics and Antagomir-31 were transfected into the cells via the
Ribo FECT TM transfection system. Then the cells were harvested to
investigate the changes of the expressions of the mRNA and proteins.
Results: We observed that LSCs LSC cultured in ESC-CM had
an increased proliferative capacity, greater serial passage capacity,
higher colony-forming efficiency (CFE) and higher levels of stem
cell associated marker than those cultured in CnT-20. Compared
with CnT-20, the level of miR-31 in ESC-CM was relative lower.
We investigated the role that miR-31 and FIH-1 play in regulating
the functional properties in LSCs. We used antagomirs (antago)
to reduce the level of miR-31 in LSCs, and a miR-31-resistant
FIH-1 to increase FIH-1 levels. Antago-31 raised FIH-1 levels and
significantly reduced p21 expressional level in LSCs compared to
irrelevant-antago treatment. The down regulation of miR-31 in LSCs
promotes the maintenance of stemness.
Conclusions: Our results demonstrate that ESC-CM regulates the
fate of LSCs partially via Notch signaling, controlled through a
miR-31/FIH-1/p21 axis. This study may have high impact and clinic
implication on the expansion of LSC in regenerative medicine,
especially for ocular surface reconstruction.
Commercial Relationships: Zhiping Liu; Xiangyin Sha, None;
Zhiqun Min, None; Huyong Zou, None; Ye Wen, None; Jing Zeng,
None
Support: NSFC Grant 81300732
Purified in vitro-expanded ABCB5-positive limbal stem cells
possess corneal regenerative capacity
Andrew Hertsenberg3, 2, Ruth Y. Lewis3, Nicholas J. Pondelis3,
Christoph Ganss6, 7, Andreas Kluth6, 7, Natasha Y. Frank1, 2,
Bruce Ksander3, 2, Markus H. Frank4, 5. 1Division of Genetics,
Brigham and Women’s Hospital, Boston, MA; 2Harvard Medical
School, Boston, MA; 3Massachusetts Eye and Ear Infirmary,
Boston, MA; 4Transplant Research Program, Boston Children’s
Hospital, Boston, MA; 5Harvard Stem Cell Institute, Boston, MA;
6
Ticeba GmbH, Heidelberg, Germany; 7Rheacell GmbH & Co. KG,
Heidelberg, Germany.
Purpose: We previously demonstrated that freshly isolated human
KRT12-negative ABCB5-positive (ABCB5+) limbal stem cells
(LSC) produce long-term (13 months) regeneration of a mature
KRT12-expressing corneal epithelium when transplanted onto
immunodeficient NSG mice with induced limbal stem cell deficiency
(LSCD). Here we examined whether purified in vitro-expanded
ABCB5+ LSC retain the capacity for corneal regeneration.
Methods: Purified in vitro-expanded human ABCB5+ LSC derived
from four separate cadaveric donors were placed into fibrin gels (500
ABCB5+ LSC / gel graft; n= 4-5 mice per group per experiment)
and subsequently grafted to immunodeficient NSG mice with
mechanically induced LSCD, through removal of the epithelium from
limbus to limbus using an Algerbrush. Negative controls were NSG
mice with induced LSCD, but treated with grafts containing the gelcarrier only and no cells. Transplants were examined weekly via slit
lamp and scored for opacity (0-4) using a standard scoring system.
Results: NSG mice with induced LSCD that received negative
control grafts containing no cells developed conjunctivalization and
opaque corneas (2.5 ± 0.67 average opacity score ± SD at 5 weeks
post transplantation). In contrast, grafts consisting of purified in
vitro-expanded ABCB5+ LSC prevented conjunctivalization and
regenerated a clear corneal epithelium 5 weeks post transplantation
(0.25 ± 0.5 average opacity score ± SD; P < 0.05 versus negative
controls by t-test).
Conclusions: Purified in vitro-expanded ABCB5+ LSC retain the
corneal regenerative capacity of freshly isolated human ABCB5+
LSC, pointing to therapeutic potential of this novel cell population
also for clinical application in treatment of human LSCD.
Commercial Relationships: Andrew Hertsenberg; Ruth Y. Lewis,
None; Nicholas J. Pondelis, None; Christoph Ganss, Ticeba
GmbH, Ticeba GmbH (I); Andreas Kluth, Ticeba GmbH;
Natasha Y. Frank, Ticeba GmbH (P); Bruce Ksander, Ticeba
GmbH (F), Ticeba GmbH (P); Markus H. Frank, Ticeba GmbH (S),
Ticeba GmbH (P)
Support: NIH 5T32EY007145-18
Development of functional cell junctions at the corneal limbus in
the embryonic chicken
James K. Kubilus, Carolina Zapater i Morales, Thomas Linsenmayer.
Integrative Physiology and Pathobiology, Tufts University School of
Medicine, Boston, MA.
Purpose: It is known that the corneal limbus contains a population
of corneal stem cells responsible for maintaining the cornea both
during health and disease. However, it is unclear how these stem
cells become established in their niche during development. We
hypothesize that the developmental expression of cell junction
proteins is necessary for the establishment of the stem cells in their
niche. It has been shown previously that the gap junction protein,
Connexin 43, is expressed in the cornea and sclera, but not in the
limbus. Here we examined the developmental expression of connexin
43 in the chicken embryo in relation to other corneal markers and
tested the potential communication between cells in the corneal
limbus.
Methods: Embryonic chicken anterior eyes from embryonic day 4
(E4) through E17 were dissected and either fixed or frozen fresh for
immunohistochemistry with antibodies against junctional proteins
connexin 43, afadin and ZO-1. Scanning electron microscopy (SEM)
was used to observe morphological changes. Cut loading of anterior
eyes from corresponding timepoints with rhodamine-dextran (MW
3000), rhodamine dextran (MW 10000), Lucifer yellow (MW 457)
and carboxyfluorescein (MW 376) was used to determine cellular
communication.
Results: Previous results using the lipophilic dye DiI, suggested that
a barrier to diffusion from the cornea to the sclera began to develop
within the limbus at approximately E8. Here, both morphological and
molecular changes consistent with this development were observed.
Beginning at E8 cells at the presumptive corneal limbus began to
change shape and level of adhesion in scanning electron microscopy.
Consistent with this, expression of connexin43 decreased in cells of
the corneal limbus. Further, cut-loading of the fluorescent dyes with a
size small enough to pass through gap junctions (rhodamine-dextran
[MW3000], carboxyfluorescein and Lucifer yellow) confirmed the
function of gap junctions within the cornea, but also showed that the
limbal cells were isolated by their lack of gap junction proteins.
Conclusions: The results suggest that the developmental regulation
of cellular junctions at the corneal-scleral limbus may have a
functional role in establishing the corneal stem cell niche through
changes in cell-cell communication. Further work is needed however,
to fully test this hypothesis.
Commercial Relationships: James K. Kubilus, None;
Carolina Zapater i Morales, None; Thomas Linsenmayer, None
Support: NIH Grant R01EY023569
PRGF-Endoret®: A new approach in the treatment of limbal
stem cell deficiency
Jesus Merayo-Lloves1, Ana Cristina Riestra1, Alvaro Meana1,
Natalia Vazquez1, Manuel Chacon1, Mairobi Persinal1, Gorka Orive2,
Eduardo Anitua2. 1Instituto Universitario Fernández-Vega, Oviedo,
Spain; 2Biotechnology Institute, Vitoria, Spain.
Purpose: The objective of the present study is to develop a complete
autologous therapy for the treatment of limbal stem cell deficiency
using PRGF® technology.
Methods: Blood collection: Plasma rich in growth factors
(PRGF) eye drop was obtained using the Endoret (PRGF) kit in
Ophthalmology (BTI Biotechnology Institute, S.L., Vitoria, Spain).
Briefly, blood was collected from human blood of healthy donors,
after informed consent, into 9mL tubes containing sodium citrate
as anticoagulant. Blood was centrifuged at 580g for 8 minutes until
blood fractionation.
F1 serum supplement: F1 fraction was used as supplement of the
culture medium. For this purpose, F1 fraction was activated with
CaCl2, incubated at 37C for one hour, and finally, the released
supernatants were collected by aspiration, aliquoted and stored at –
80C until use.
F2 membrane: F2 fraction was used for the development of scaffolds.
Briefly, 5mL of F2 fraction was collected, activated with CaCl2 and
incubated at 37C. Once a gel was formed, it was flattened under
mechanical pressure obtaining a 100µm membrane.
Cellular culture: Corneoscleral tissue was attained from a local
eye bank after penetrant-keratoplasty surgery. Human limbal stem
cells (hLSC) were obtained from explants of 2-3mm in diameter
of the limbal region and cultured in DMEM/F12 supplemented
with 100U/mL penicillin, 0.1mg/mL streptomycin and 10% F1
serum supplement. When the culture reached confluence, cells were
seeded onto F2 fraction membrane, cultured and fixed with ice-cold
methanol when confluent.
Cellular analysis: Cellular growth was assessed by phase
contrast microscopy and scanning electron microscopy (SEM).
Immunocytochemistry for p63 and cytokeratin was also performed in
order to check their immunological markers.
Results: Using F2 serum fraction we were able to achieve a
manageable membrane of 100µm. This membrane was able to
support cellular growth of hLSC, which were cultured with F1
serum fraction as an only supplement of the culture media. In these
conditions, hLSC displays their typical polyhedral morphology while
expressing their characteristic cellular markers, p63 and cytokeratin.
Conclusions: PRGF-Endoret® technology provides an autologous
environment for the isolation of hLSC as well as an autologous
membrane for their growth. This strategy allows the development of a
complete autologous cell therapy for the treatment of limbal stem cell
deficiency.
Commercial Relationships: Jesus Merayo-Lloves, None; Ana
Cristina Riestra, None; Alvaro Meana, None; Natalia Vazquez,
None; Manuel Chacon, None; Mairobi Persinal, None;
Gorka Orive, None; Eduardo Anitua, None
Support: Telefonica // retos colaboración (RTC-2014-2375-1)
The Role of Thrombomodulin in Epithelial Keratopathy
Yi-Hsun Huang. National Cheng Kung University Hospital, Tainan,
Taiwan.
Purpose: Thrombomodulin (TM) improved skin wound healing
process. However, no definite data related to corneal epithelial
wound healing process were proved till now. We aim to explore the
expression of TM in wound healing process, epithelial keratopathy
and to investigate whether recombinant TM1 domain (rTMD1) has
therapeutic potential in such conditions.
Methods: TM localization and expression in the normal and diabetic
corneas were examined by immunofluorescence staining. TM
expression after injury was also studied. The effect of rTMD1 on
corneal wound healing was evaluated by in vitro and in vivo assays.
Results: TM was expressed in the cornea in normal and STZinduced diabetic mice. TM expression increased in the early phase
of wound healing and decreased after wound recovery. In the in
vitro study, platelet-derived growth factor-BB (PDGF-BB) induced
TM expression in murine corneal epithelial cells by mediating E26
transformation-specific sequence-1 (Ets-1) via the mammalian target
of rapamycin (mTOR) signaling pathway in normal mice. In diabetic
mice, HMGB1 might play a role in the corneal epithelial wound
healing process. The administration of rTMD1 increased the rate of
corneal epithelial wound healing.
Conclusions: As our previous study showed, TM expression in
corneal epithelium was modulated during the corneal wound healing
process. In DM patients, rTMD1 might have therapeutic potential in
corneal injury.
Commercial Relationships: Yi-Hsun Huang, None
Support: NCKUH-10406017
Kinetics of extracellular matrix (ECM) protein release from
cornea following epithelial injury and its relevance to wound
repair
SOMSHUVRA BHATTACHARYA, Gudiseva Chandrasekher.
Pharmaceutical Sciences, South Dakota State University, Brookings,
SD.
Purpose: Corneal epithelial injury induces significant changes in
cellular physiology of stromal layer that results in the release of
growth promoting factors to facilitate the healing. Wound repair is
a complex process that requires the participation of different ECM
proteins for cell adhesion, migration and proliferation. To attribute
a distinct function for different ECM proteins during epithelial
regeneration we analyzed the pattern of their release after epithelial
debridement.
Methods: Porcine corneas subjected to injury by complete epithelial
debridement were maintained in organ culture (DMEM/F12
medium at 37 oC for 0- 60 hours). The culture medium was removed
periodically and replaced with fresh medium. The presence of
different ECM proteins in the medium was analyzed by SDS-PAGE/
immunoblotting. A preparation of epithelial primary cultures and
ECMs (collagen I and fibronectin), and a pectin-Ca based scaffold
embedded with cornea primary epithelial cultures and ECMs
were evaluated for their ability to regenerate epithelial layer on
deepithelialized corneas and in cell culture respectively.
Results: Presence of collagen I, collagen IV and fibronectin was
identified in the medium in which the corneas were incubated. As
compared to uninjured corneas, deepithelialized corneas released
significantly higher amount of ECM proteins. Rate of release of these
proteins varied with time. While collagen I release mostly remained
steady throughout the experimental conditions, fibronectin release
was higher in first four hours of incubation and collagen IV levels
increased in 8-12 hours. Release of all three proteins decreased
gradually, but sustained beyond 60 hours. Topical administration
of a mixture of epithelial cells and ECM proteins promoted the
regeneration of epithelium on epithelial debrided cornea surface. A
scaffold embedded with ECM proteins and epithelial cells produced
the growth of epithelial layer in cell culture.
Conclusions: Fibronectin released early from injured cornea may
form a provisional basement membrane to initiate epithelial adhesion.
Delayed release of collagen IV could facilitate the formation of
permanent basement membrane. Prolonged release of ECM proteins
indicates their necessity for epithelial cell regeneration. Engineering
of ECM-based therapeutic devices for regeneration of damaged
ocular surface is worthy of consideration.
Commercial Relationships: SOMSHUVRA BHATTACHARYA,
None; Gudiseva Chandrasekher, None
Support: South Dakota State University Scholarly activity and
Excellence Fund, Dept. of Pharmaceutical Sciences SDSU
Blueberry Component Pterostilbene Protects Corneal Epithelial
Cells from Inflammatory and oxidative stress
Ding Chen1, 2, Jin Li1, 2, Ruzhi Deng1, 2, Xia Hua1, Lili Zhang1,
Stephen C. Pflugfelder1, De-Quan Li1. 1Ocular Surface Center, Cullen
Eye Institute, Department of Ophthalmology, Baylor College of
Medicine, Houston, TX; 2School of Optometry and Ophthalmology,
Wenzhou Medical University, Wenzhou, China.
Purpose: Pterostilbene (PS), a naturally dietary compound of
blueberries, has been proven to be an anti-inflammatory and antioxidative agent. However, there is no study on its effect on ocular
diseases, such as dry eye. This study was to explore potential
protective effects of PS on dry eye using an in vitro culture model
of human corneal epithelial cells (HCECs) exposed to hyperosmotic
medium.
Methods: Primary HCECs were cultured from fresh donor limbal
explants. Hyperosmolarity model was established by switching
HCECs from isosmotic (312 mOsm) to hyperosmotic medium (450
mOsm) alone or in the presence of different concentration (5-20μM)
of PS for 4-24h. Gene expression was detected by RT-qPCR; and
protein production or activity was evaluated by ELISA, zymography,
Western blotting and immunostaining. Reactive oxygen species
(ROS) production was measured using a DCFDA kit.
Results: The addition of PS significantly reduced the expression
of pro-inflammatory mediators, TNF-α, IL-1β, IL-6, MMP-2
and MMP-9 in HCECs exposed to hyperosmotic medium. Pretreatment with PS (5 to 20µM) suppressed ROS overproduction in
a dose-dependent manner. Additionally, PS significantly decreased
the levels of oxidative damage biomarkers, malondialdehyde
(MDA), 4-hydroxynonenal (4-HNE), aconitase-2 and
8-hydroxydeoxyguanosine (8-OHdG). Importantly, PS was found
to rebalance homeostasis between oxygenases and anti-oxidative
enzymes by decreasing cyclooxygenase 2 (COX2) expression and
restoring the activity of antioxidant enzymes, super oxide dismutase 1
(SOD1) and peroxiredoxin-4 (PRDX4) during hyperosmotic stress.
Conclusions: Our findings demonstrate for the first time that
blueberry component pterostilbene protects the human cornea
from hyperosmolarity-induced inflammation and oxidative stress,
suggesting the protective potential to dry eye disease.
Commercial Relationships: Ding Chen, None; Jin Li; Ruzhi Deng,
None; Xia Hua, None; Lili Zhang, None; Stephen C. Pflugfelder,
None; De-Quan Li, None
Mixed Platelet-Rich Plasma Eye Drops for the Treatment of
Persistent Corneal Epithelial Defects
Sahar Balagholi1, 2, Behnam Ranjbar2, Siamak Delfazaye
Baher3, Alireza Baradaran-Rafiei1, Mozhgan Rezaeikanavi1,
Shaban Alizadeh2. 1Ocular Tissue Engineering Research Center,
Shahid Beheshti University of Medical Sciences, Tehran, Iran (the
Islamic Republic of); 2Department of Hematology, School of Allied
Medicine, Tehran University of Medical Sciences, Tehran, Iran (the
Islamic Republic of); 3Ophthalmic Research Center, Shahid Beheshti
University of Medical Sciences, Tehran, Iran (the Islamic Republic
of).
Purpose: To investigate the effects of mixed platelet-rich plasma
(mix PRP) eye drop on the healing of persistent corneal epithelial
defects.
Methods: Mixed PRP eye drops were prepared with a specific ratio
of autologous platelet-rich plasma and cryoprecipitate, aliquoted to
sterile multidose vials. Fifty patients with persistent corneal epithelial
defects were treated with mix PRP eye drops four times a day. The
patients were categorized into post-surgical, post-transplantation,
dry eyes, chemical burns and miscellaneous subgroups. Slitlamp
biomicroscopic examinations together with photoslit photography
were performed at the baseline and weekly until complete healing.
Mean duration of treatment between subgroups was analyzed by
ANOVA test. Moreover, mean duration of corneal defect before and
after initiation of the treatment was compared by paired t-test.
Results: Mean duration of treatment in the post-surgical subgroup
(2.5 ± 1.8 wk) was not significantly different from the posttransplantation (3.2 ± 2.3 wk), dry eyes (2.0 ± 0.0 wk), chemical
burns (2.1 ± 0.8 wk), and miscellaneous (1.8 ± 1.2 wk) subgroups.
Mean duration of corneal defect after initiation of treatment (2.65 ±
1.84 w) was significantly less than what before the treatment (5.35
± 1.06 wk) (P value < 0.001); i.e. mixed PRP eye drops led to rapid
healing of persistent corneal epithelial defects in all subgroups.
Conclusions: Treatment with autologous mixed PRP eye drops seems
to be an effective and reliable approach that accelerates epithelial
would healing in cases with persistent corneal epithelial defects of
various etiologies.
Commercial Relationships: Sahar Balagholi, None;
Behnam Ranjbar; Siamak Delfazaye Baher, None;
Alireza Baradaran-Rafiei, None; Mozhgan Rezaeikanavi, None;
Shaban Alizadeh, None
Improved Ocular Surface Discomfort Index and Ocular
Discomfort Index scores with topical autologous serum
Catherine Croghan, Deepa Anijeet. Tennent Institute of
Ophthalmology, Gartnavel General Hospital, Glasgow, United
Kingdom.
Purpose: Autologous serum drops have previously been shown
to improve clinical and laboratory indicators, with improved
Schirmer’s test, reduced tear film debris, increased density of goblet
and epithelial cells, and reduced apoptotic cells. The aim of this
retrospective case series was to investigate the effect of autologous
serum drops on frequency of lubricant application, and any change
in the Ocular Surface Discomfort Index (OSDI) and the Ocular
Discomfort Index (ODI) after treatment
Methods: A retrospective case series is presented of 17 patients using
autologous serum topically, from 07/05/11. Autologous serum had
been commenced due to graft versus host disease (n=6), Sjögren’s
syndrome (n=3), ocular cicatricial pemphigoid (n=2), stem cell failure
(n=2), trauma to the lacrimal gland (n=1), ectodermal dysplasia
(n=1), Stevens-Johnson syndrome (n=1), and neurotrophic cornea
due to diabetes mellitus (n=1). There were 8 males, and 9 females.
The mean age was 47(±14) years. Patients used autologous serum
drops for a mean 16 (±13) months. The outcome measures were
frequency of use of lubricants, OSDI and ODI scores. The outcome
measures were assessed at two time points, prior to introduction of
autologous serum drops and also at most recent review. The OSDI
and ODI questionnaires were completed retrospectively by telephone
interview. The Student’s t test was used for statistical analysis
Results: Patients instilled a mean 9 (±4) autologous serum drops
per day. Prior to autologous serum, patients instilled a mean 12
(±5) drops of lubricants per day. This reduced after commencing
autologus serum to a mean 3 (±4) drops of lubricant per day. The
OSDI score reduced from a mean 85 to 49 after commencement
of autologous serum. The mean reduction of the OSDI scare was
36 (mean±standard deviation: 36±19, 95% confidence interval ±9,
p= 0.0000006). The ODI score was a mean 55 prior to autologus
serum and 39 after. The mean reduction of the ODI score was 16
(mean± standard deviation: 16±8, 95% confidence interval ±4, p=
0.0000003). 3 patients discontinued use of autologous serum; the
reasons identified in each case were evisceration, drops stinging on
application, and the process being inconvenient
Conclusions: Autologous serum drops are a safe and effective
management option. This is demonstrated by reduced frequency of
lubrication, and reduced OSDI and ODI scores
Commercial Relationships: Catherine Croghan, None;
Deepa Anijeet, None
Cryopreserved Amniotic Membrane After Epithelial
Debridement for Recurrent Corneal Erosion
Scott G. Hauswirth1, Milton M. Hom2. 1Ophthalmology & Medicine,
Minnesota Eye Consultants PA, Blaine, MN; 2Private Practice, Azusa,
CA.
Purpose: Recurrent corneal erosion is a common condition in
clinical practice. Cryopreserved amnion tissue (CAM) is increasingly
used in clinical ophthalmic practice for a variety of conditions. At
present, no comparative studies examining outcomes for treatment of
recurrent corneal erosion (RCE) utilizing bandage contact lens (BCL)
versus CAM exist. This case series attempts to examine whether
the recurrence rate of RCE treated by bandage contact lens alone
(BCLA), epithelial debridement plus BCL (ED+BCL), or epithelial
debridement plus CAM (ED+CAM) differs.
Methods: 44 eyes of 44 patients treated for RCE at a single site were
retrospectively reviewed. Patients with less than 3 months follow
up or who received treatment methods other than those studied were
excluded. Outcomes were examined between BCLA group (n=5,
males=3, mean age=51.6 years), ED+BCL group (n=5, males=1,
mean age=48.2) and ED+CAM group (n=6, males=0, average
age=60.33 years) for incidence of recurrence (defined as patient
description of an incident of pain, or visible epithelial slough on
examination), and post-treatment haze.
Results: In the BCLA group, average follow up period was 37.6mo
(range 8–84mo). Recurrence occurred in 4 patients (80%). Average
time to recurrence was 2.44mo (range 0.25-8mo). In the ED+BCL
group, average follow up period was 41.2mo (range 34-50mo).
Recurrence occurred in 2 patients (40%), with average time of
11.25mo (range 0.5-22mo). In the ED+CAM group, average followup period was 7.5mo (range 3–13mo). Although the follow up period
in the ED+CAM was shorter, no recurrence occurred, and was
comparable to mean time of recurrence across all groups (5.375mo).
Corneal haze was noted in one patient in the ED+BCL group (20%)
and one patient in the ED+CAM group (16.6%).
Conclusions: Compared to bandage contact lens alone, ED+BCL
and ED+CAM reduced rate of recurrence. No patients in the
ED+CAM group experienced signs or symptoms of recurrent
erosion during their follow up period. Based on our results, addition
of cryopreserved amniotic membrane to epithelial debridement
enhanced treatment and reduced recurrence of RCE. Incidence of
haze was similar between ED+BCL and ED+CAM groups. Further
studies using larger numbers of patients are warranted.
Commercial Relationships: Scott G. Hauswirth, BioTissue
(C), TearScience (C), Bausch+Lomb (C), Shire (C), Allergan (C);
Milton M. Hom
Measurement of Corneal Epithelial Permeability to Fluorescein
in Humans by Sequential Multi-Drop Method
Katelyn Keefer1, Sanjay A. Mahadik2, Arushi Goyal2,
Ramya Ravindran2, Prema Padmanabhan2, Sudhir Rachapalli2,
Sudhir Ranganath3, Uday B. Kompella4, Sangly P. Srinivas1. 1Indiana
University, Bloomington, IN; 2Sankara Nethralaya, Chennai, India;
3
Siddaganga Institute of Technology, Tumkur, India; 4University of
Colorado, Denver, CO.
Purpose: Fluorescein permeability (P) of the corneal epithelium is
affected in dry eye disease, aging, and diabetes. P is measured by
variations of the “fluorometric single-drop” method. This approach
suffers from poor repeatability as the measurements are influenced
by inner filter effects (IFE) and sensitivity at higher and lower
concentrations of the dye, respectively. The objective of this study,
therefore, is to establish an improved method for measurement of P
based on sequential instillation of topical fluorescein.
Methods: First, one drop of 0.35% fluorescein is instilled and its
subsequent dynamics in the tears is followed by a custom-built
ocular fluorometer. Next, 2 drops of 2% of fluorescein are instilled
10 min apart. 15 minutes later, the ocular surface is washed. Finally,
fluorescence in the stroma (Fs) is measured 4 times. Assuming
negligible loss into a/c, we derived an equation for permeability: P =
(Q * Fs)/ (2*AUC); Q is the stromal thickness and AUC = (2/0.35) *
F0/ke. F0 and ke are estimated based on fluorescence vs. time profile
following the first drop of 0.35% fluorescein. Proof-of-principle of
the different aspects of the approach has been investigated in a cohort
of healthy volunteers (n >10).
Results: The fluorescein dynamics in the tears following 0.35% drop
could be followed with a high S/N ratio, without IFE, and without
overloading the fluorometer (n>10). Analysis of the decay profile
showed expected inter-individual as well as drop-to-drop variability
in ke and F0. At 15 min following 0.35% drop, Fs was close to the
measured background. After multiple drops of the dye at 2%, Fs
increased to at least 10-20x the background and could be measured
very accurately. Based on average values of ke and F0 and assuming
stromal thickness of 475 µm, the calculated range of P varied from
5-15 nm/sec (n=5).
Conclusions: The inherent problem of IFE limits the use of
fluorescein at high concentrations. Accordingly, we chose the low
concentration drop (i.e., 0.35%) to circumvent IFE and determine
fluorescein dynamics in the tears. The high concentration drops
(i.e., 2%) surmounted the problem of poor sensitivity and allowed
Fs measurements at high precision. The measured values of P, albeit
much higher than previous reports, concurs with permeability of
many solutes reported for the corneal epithelium.
Protocol for multi-drop approach to determine corneal epithelial
permeability
Commercial Relationships: Katelyn Keefer, None;
Sanjay A. Mahadik, None; arushi goyal; Ramya Ravindran,
None; Prema Padmanabhan, None; Sudhir Rachapalli,
None; sudhir ranganath, None; Uday B. Kompella, None;
Sangly P. Srinivas, None
Support: Obama Singh Initiative Award
Silk fibroin protein enhances both in vitro and in vivo corneal
epithelial wound healing through increased cell migration,
adhesion, and proliferation
Mark Rosenblatt1, 2, Waleed Abdel-Naby2, Brigette Cole2, Jingbo Liu2,
Aihong Liu2, Pengxia Wan2, Brian Lawrence3, Victor H. Guaiquil1, 2,
Ryan Schreiner2, Enrique J. Rodriguez-Boulan2. 1Department
of Ophthalmology and Visual Sciences, University of Illinois at
Chicago, Chicago, IL; 2Department of Ophthalmology, Weill Cornell
Medical College, New York, NY; 3Silk Tears, Inc., Plymouth, MN.
Purpose: Corneal epithelial wound healing involves a set of complex
biological processes that work to restore the epithelium after injury.
Biomaterials derived from Bombyx mori silk fibroin have been used
in therapeutic applications to promote wound healing and tissue
regeneration. Our study evaluated the effect of silk fibroin, in its
solution form, on corneal epithelial cell behavior and regeneration in
vitro, and corneal epithelial wound healing in an animal model.
Methods: We used human corneal limbal epithelial cells (HCLE)
to evaluate if silk fibroin solution induces a significant effect
on epithelial cell adhesion, migration and proliferation, and the
concomitant effect on epithelial cell recover after injury. To study
cellular adhesion, we utilized a parallel plate flow chamber and
analyzed focal adhesion protein by immunofluorescence staining.
Migration of epithelial cells after a scratch injury was quantitatively
analyzed using time lapse imaging and the spreading of epithelial
cells in response to treatment with silk fibroin solution was quantified
using phase contrast imaging. Cell proliferation was evaluated using
an MTT colorimetric assay as well as flow-cytometric cell cycle
analysis. The effect of silk fibroin solution in promoting corneal
epithelial wound healing in vivo, was analyzed in a rabbit corneal
abrasion model.
Results: Our in vitro results using HCLE cells demosntarted that
silk fibroin solution enhanced cell matric focal adhesions by over
95% compasred to control. Similarly, we observed a 50% increase in
epithelial cell migration and a 60% enhancement in cell proliferation
when cells were treated with silk fibroin solution. Epithelial wound
closure showed a 2 fold increase in healing rate by 10 hours with
0.4% wt./vol. silk fibroin and a 30% improvement in wound closure
by 15 hours. HCLE cells that were mitotically inhibited showed
a 30% increase in wound closure when treated with silk fibroin
solution. In our in vivo model, silk fibroin solution accelerated the
recovery of the injured corneal epithelium.
Conclusions: Our findings indicate that silk fibroin solution induces
major rearrangements on the cell cytoskeleton that may contribute to
enhanced cell adhesion, migration and proliferation, leading to faster
re-epithelialization of corneal wounds.
Commercial Relationships: Mark Rosenblatt; Waleed AbdelNaby, Silk Tears, Inc. (F); Brigette Cole, None; Jingbo Liu, None;
Aihong Liu, None; Pengxia Wan, None; Brian Lawrence, Silk
Tears, Inc.; Victor H. Guaiquil, None; Ryan Schreiner, None;
Enrique J. Rodriguez-Boulan, None
Support: Research to Prevent Blindess
A thin rigid contact lens used in vitreous-retinal surgery for
corneal protection
Songtao Yuan, Zizhong Hu, Ping Xie, Qinghuai Liu. Department
of Ophthalmology, First Affiliated Hospital of Nanjing Medical
University, Nanjing, China.
Purpose: Corneal epithelial dryness or damage is more concerned
in complicated proliferative diabetic retinopathy (PDR) surgery if
using noncontact wide-angle viewing system. We designed a rigid
contact lens to be used during surgery and performed a prospective,
randomized, comparative study to analyze its protection of corneal
epithelia.
Methods: A thin and lightweight rigid contact lens was designed and
constructed. The impact of the contact lens on the visualized fundus
range was evaluated using a concrete eye model. Eighty two eyes of
74 patients with severe PDR were randomized to either the contact
lens (CL) group, viscoelastic group (VIS), or balanced salt solution
(BSS) group. Surgery time and corneal fluorescein staining score
(FSS) postoperatively were mainly measured using one way analysis
of variance (ANOVA) test.
Results: In the eye model, a larger area of fundus was visualized
with the use of our contact lens under the 128D (>80°) and 60D (45°)
Resight lens compared to the 128D (76°) and 60D (42°) Resight
lens alone. The mean surgery time was 51.36±8.06min,50.89±8
.26min,and 55.46±9.14 in CL, VIS, and BSS group respectively
(F=0.493,p=0.105). In four eyes of the BSS group, corneal epithelial
layer was peeled because the serious dryness of the cornea could
not maintain a clear fundus image. The FSS in BSS group was
markedly higher than that of CL and VIS group 6 hours (7.22±3.81,
1.43±1.62, and 1.70±1.96 respectively, F=36.48, p<0.001), 1
day (5.33±3.40, 1.11±1.42 and 1.41±1.78 respectively, F=23.83,
p<0.001), 3 days (3.33±2.02, 0.82±1.06 and 0.93±1.27 respectively;
F=20.28, p<0.001), and 7 days (1.67±1.64, 0.50±0.80, and 0.63±0.93
respectively, F=6.865,p=0.002) postoperatively. There was no
statistical significance of the FSS score between CL and Vis group at
each follow-up endpoint.
Conclusions: The contact lens we designed can slightly enlarger the
visible fundus range and efficiently protect corneal epithelia from
damage during vitrectomy in PDR patients.
Contact lens and the eye model for evaluation of visualized fundus
range
Visualized fundus with the use of our contact lens under the 60D
(45°) and 128D (>80°) Resight lens compared to the 60D (42°) and
128D (76°) Resight lens alone under 4mm pupil.
Commercial Relationships: Songtao Yuan, None; Zizhong Hu,
None; Ping Xie, None; Qinghuai Liu, None
Support: 2013CB967500;BL2014089
Clinical Trial: http://58.213.51.73:8088/, 2014-SR-030
Influence of contact lens wearing on whole corneal thickness
and corneal epithelial thickness measured with Fourier-domain
optical coherence tomography
Joanna Wasielica-Poslednik1, Marcel S. Sievers1, Walter Lisch1,
Veronika Weyer2, Norbert Pfeiffer1, Adrian Gericke1. 1Dept. of
Ophthalmology, University Medical Center of the Johannes
Gutenberg-University Mainz, Mainz, Germany; 2Institute for Medical
Biostatistics, Epidemiology and Informatics (IMBEI), University
Medical Center of the Johannes Gutenberg-University Mainz, Mainz,
Germany.
Purpose: To investigate the effect of contact lens use on whole
corneal thickness (CT) and corneal epithelial thickness (ET)
measured with Fourier-domain optical coherence tomography (FDOCT, RTVue).
Methods: Both eyes of 17 soft contact lens (SCL) wearers, 9 hard
contact lens (HCL) wearers and 31 age- and sex-matched controls
were included in this prospective study. CT and ET were measured
with FD-OCT in 9 areas: corneal vertex, 4 paracentral and 4
peripheral zones in control subjects and in CL-wearers immediately
after discontinuing of CL. The second measurement was performed
in controls and CL-wearers after five days of CL-discontinuation.
At each visit, refraction error, best-corrected visual acuity, slit-lamp
examination and Schirmer test were performed in both eyes of all
subjects. Statistical analyses were conducted using ANOVA, MannWhitney test, t-test and Wilcoxon test.
Results: ET differed relevantly in vertex and in paracentral zones
between the individual groups (HCL < SCL < controls, p<0.05) at
baseline. After five days of CL restriction, an increase of ET in vertex
and in paracentral zones was noted in the HCL-wearers (p<0.05), but
not in the SCL-wearers and in the control group. CT did not differ
relevantly between the study groups at baseline [HCL > control >
SCL (p>0.05)]. Five days of contact lens restriction had no relevant
influence on CT, BCVA, refraction error and Schirmer test.
Conclusions: ET was relevantly thinner in vertex and in corneal
paracentral zones in CL wearers compared to controls. A five day
discontinuation of CL wearing led to an increase in ET, which was
relevant in HCL-wearers.
Commercial Relationships: Joanna Wasielica-Poslednik, None;
Marcel S. Sievers, None; Walter Lisch, None; Veronika Weyer,
None; Norbert Pfeiffer, None; Adrian Gericke, None
Interleukin-32 induced thymic stromal lymphopoietin plays a
critical role in the inflammatory response of Human Corneal
Epithelium
Jing Lin, Guiqiu Zhao, Qian Wang. The Affiliated Hospital of
Qingdao University, Qingdao, China.
Purpose: Interleukin (IL)-32 is a newly discovered cytokine and
has been associated with a variety of inflammatory diseases. Thymic
stromal lymphopoietin (TSLP) plays important roles in mucosal
epithelial cells, especially in allergy-induced inflammation, through
the TSLP-TSLPR(Thymic stromal lymphopoietin receptor) signaling
pathway. However, the relationship between IL-32 and TSLP in the
ocular surface remains unclear. This study sought to identify the
relationship and function of IL-32 and TSLP in regulating the proinflammatory cytokine production in corneal epithelial cells.
Methods: Human corneal tissues and cultured primary human
corneal epithelial cells (HCECs) were treated with different IL-32
concentrations, in the presence or absence of various inhibitors
to evaluate TSLP expression and localization and the signaling
pathways involved in regulating pro-inflammatory cytokine
production. TSLP mRNA expression was determined by reverse
transcription and real time PCR, and protein production was
measured by ELISA and immunohistochemical staining.
Results: TSLP protein expression was examined in donor
corneal epithelium. IL-32 significantly stimulated TSLP and proinflammatory cytokines (TNFα and IL-6) production in HCECs at
both mRNA and protein levels. The production of pro-inflammatory
mediators by IL-32 was increased by recombinant TSLP protein.
Interestingly, the NF-κB inhibitor quinazoline and the caspase-1
inhibitor VX-765 suppressed IL-32 induced pro-inflammatory
cytokines production (TNFα and IL-6).
Conclusions: These findings demonstrate that IL-32 and IL-32
-induced TSLP are important cytokines involved in inflammatory
responses through caspase-1 and NF-κB signaling pathways, in
human corneal epithelium, suggesting novel molecular targets in the
inflammatory diseases of the ocular surface.
Commercial Relationships: Jing Lin, None; Guiqiu Zhao, None;
Qian Wang, None
Support: National Natural Science Foundation of China (No.
81170825)
Functionality of a liposome-based anti-inflammatory formulation
in an in vitro corneal inflammation model
Laura Soriano-Romani1, 2, Marta Vicario-de-la-Torre3, Mario CrespoMoral1, Antonio Lopez-Garcia1, 2, Rocio Herrero-Vanrell3, Irene
T. Molina-Martínez3, Yolanda Diebold1, 2. 1Ocular Surface GroupIOBA, University of Valladolid, Valladolid, Spain; 2Biomedical
Research Networking Center on Bioengineering, Biomaterials and
Nanomedicine (CIBER-BBN), Madrid, Spain; 3Department of
Pharmacy and Pharmaceutical Technology, Faculty of Pharmacy,
Complutense University, Madrid, Spain.
Purpose: Unpreserved liposomal topical formulation used as
artificial tears was loaded with the anti-inflammatory agent
medroxyprogesterone acetate (MPA). Our aim was to characterize
MPA-loaded liposomes (MPA-LP), study its uptake by corneal
epithelial cells, and determine its functionality in an in vitro corneal
inflammation model.
Methods: Liposomes (LP) composed of phosphatidylcholine,
cholesterol, vitamin E, and MPA at a molar ratio 13:3:0.2:0.3
were prepared by solvent evaporation technique and labeled with
coumarin-6 (C6-LP) for uptake experiments. LP characterization
included diameter size, pH, osmolarity, and viscosity. Drug
encapsulation efficiency was also calculated. Human corneal
epithelial (HCE) cells were used to in vitro study MPA-LP-induced
effects after 60 min of exposure. Blank LP and a MPA reference
formulation (Colicursi Medrivas, Alcon Cusi S.A.) were used as
controls. LP cytotoxicity was analyzed by the XTT assay. C6-LP
uptake by HCE cells was analyzed immediately, 24, 48, and 72h
after the exposure period. Changes in protein expression and nuclear
translocation of MPA glucocorticoid and progesterone receptors were
determined by Western blotting and immunofluorescence. Changes
in cell proliferation were measured by the AlamarBlue assay. Also,
TNFα-stimulated cells (inflamed) were exposed to MPA-LP and
changes in IL-6 and IL-8 production were analyzed by ELISA.
Results: LP showed adequate physicochemical properties for topical
ophthalmic formulations and MPA encapsulation efficiency was
close to 94%. HCE cells showed LP-associated fluorescence at 24,
48, and 72h after the exposure period without any cytotoxic effect.
The protein expression and nuclear translocation of progesterone
receptor increased in MPA-LP-exposed cells compared to blank LPexposed cells, indicating MPA-LP uptake by HCE cells. Moreover,
MPA-LP showed anti-proliferative and anti-inflammatory effect by
reducing cell proliferation rate and IL-6 and IL-8 production 48h
after the exposure period. None of these effects were shown in blank
LP-exposed cells, and the reference formulation only reduced IL-6
production.
Conclusions: The LP-based formulation used to replenish the
lipids of the tear film may also be used as an anti-inflammatory
drug delivery system for the treatment of inflammatory processes
associated to ocular surface diseases.
Commercial Relationships: Laura Soriano-Romani, None;
Marta Vicario-de-la-Torre, None; Mario Crespo-Moral, None;
Antonio Lopez-Garcia, None; Rocio Herrero-Vanrell, None; Irene
T. Molina-Martínez, None; Yolanda Diebold, None
Support: FEDER-CICYT MAT2013-47501-C2-1-R, Regional JCyL
Scholarship/European Social Fund Program (VA098-12) Research
Group UCM 920415 (GR/14), RETICS-RD07/0062/0013, FEDERCICYT FIS-PI10/00645 and PI10/00993 (Ministry of Health, Spain).
Assessment of corneal changes associated with topical
antiglaucoma therapy using in vivo confocal microscopy
Elmira Baghdasaryan2, Tudor Tepelus2, Laura Vickers1, Ping Huang2,
Srinivas R. Sadda2, 1, Olivia L. Lee2, 1. 1Ophthalmology, Doheny Eye
Center, University of California Los Angeles, Los Angeles, CA;
2
Doheny Image Reading Center, Doheny Eye Institute, Los Angeles,
CA.
Purpose: Ocular surface disease (OSD) is prevalent amongst eyes
treated with chronic topical antiglaucoma (A/G) therapy. The purpose
of this study was to evaluate the density of corneal epithelial cells,
presence of epithelial dendritic cells (DC) and sub-basal nerve plexus
characteristics in eyes on topical A/G therapy, based on imaging by in
vivo confocal microscopy (IVCM).
Methods: Central corneal images were prospectively captured from
16 eyes of 11 patients on topical A/G therapy (>6 months) and 10
normal control eyes, using IVCM (HRT III RCM, Heildelberg,
Germany). Demographic data was collected, as well information
on the types and duration A/G therapy. In addition, ocular surface
disease index (OSDI) score, density of epithelial wing cells (WC)
and basal cells (BC), sub-basal nerve features (density, tortuosity and
reflectivity), and presence of DC were all assessed and recorded by
trained reading center graders.
Results: Mean OSDI score was 6.36 ± 6.07 in controls and 30.20
± 33.75 in glaucoma patients on topical A/G therapy (p=0.05).
Nerve fiber density and reflectivity of the sub-basal plexus were
significantly lower in eyes on A/G topical therapy as compared with
controls (1869.42 ± 785.40 μm/ frame versus 2724.86 ± 687.06 μm/
frame, p-value =0.008 and 2.83 ±0.83 versus 3.50±0.55, p-value
= 0.02). Nerve tortuosity was significantly higher in eyes on A/G
drops group as compared to controls (2.87 ± 0.57 versus 2.13 ±0.48,
p-value=0.003). DC displayed significantly increased density in
eyes treated with A/G drops than in controls (72.94± 61.74 cells/
mm2 versus 27.53 ±5.58 cells/mm2, p-value =0.01). WC and BC
densities were both lower in glaucomatous eyes on A/G drops group
as compared with controls; the difference in WC density was not
statistically significant (5658.94±654.88 cells/mm2 vs. 5812.62
±772.74 cells/mm2, p-value = 0.6), while the decrease in BC density
in A/G treatment group was statistically significant (6442.75 ±692.55
cells/mm2 vs. 8479.20 ± 833.50 cells/mm2, p-value < 0.001).
Conclusions: Our study revealed significant microstructural changes
in the corneas of eyes treated with topical A/G therapy that is
associated with ocular surface disease symptoms. IVCM may be a
useful tool for the assessment of the ocular surface toxicity associated
with glaucoma topical therapy
Commercial Relationships: Elmira Baghdasaryan, None;
Tudor Tepelus; Laura Vickers, None; Ping Huang, None;
Srinivas R. Sadda, NOVARTIS (C), ICONIC (C), STEM CELLS
INC. (C), GENENTECH (C), BAYER (C), GENENTECH (F),
OPTOS (F), REGENERON (C), ALLERGAN (F), Carl Zeiss
Meditec (F), ALLERGAN (C), AVALANCHE (C), OPTOS (C),
THROMBOGENICS (C); Olivia L. Lee, ALLERGAN (C),
ALLERGAN (F)
Purpose: We evaluated eyelid tension using a novel eyelid
tensiometer and quantified the effect of reduced eyelid tension
by injecting botulinum toxin A to treat severe intractable dry
eye (DE) combined with filamentary keratitis or superior limbic
keratoconjunctivitis (SLK).
Methods: Forty eyes from 20 patients with intractable severe
DE (level 3 or 4) and filamentary keratitis or SLK were included.
Patients were subcutaneously injected with botulinum toxin A (2.5
units/0.1 mL per site) in each medial and lateral pretarsal orbicularis
muscle of the upper eyelid. After verifying intra- and interobserver
reliability, static and dynamic eyelid tension forces were measured
using a newly developed eyelid tensiometer before and at one and
three months after injection. At the same time, the Schirmer I test was
performed, and tear film break-up time (tBUT) and corneal staining
score (CSS) were estimated as clinical outcome measurements.
Results: The intra- and interobserver reliability of the invented eyelid
tensiometer were excellent, with intraclass correlation coefficients
ranging from 0.988 to 0.991 and from 0.988 to 0.992, respectively.
The mean patient age was 57.95 +- 3.37 years. Tear secretion,
tBUT, and CSS, as well as corneal filaments and superior bulbar
conjunctival erosions in SLK improved, and significant reduction in
both static and dynamic lid tension was observed at one (1.026 +0.013 N and 1.162 +- 0.027 N, respectively) and three months (1.068
+- 0.011 N and 1.286 +- 0.044 N, respectively) compared to baseline
before botulinum toxin A injection (1.102 +- 0.016 N and 1.456 +0.058 N, respectively).
Conclusions: The change in eyelid tension after botulinum toxin
A injection to treat intractable severe DE combined with SLK or
filamentary keratitis was quantitatively measured using the novel
eyelid tensiometer. Eyelid tension reduction can be a clinically useful
treatment for the management of intractable severe DE with elevated
eyelid tension.
A newly developed eyelid tensiometer consisting of (A) a speculum
and a voltage meter. (B) The sensor and the magnet are labeled on
each arm of the speculum. Counterweights are used to conversion
formula convert millivolts to Newton.
Eyelid tension reduction for treating intractable dry eyes and
quantification using an eyelid tensiometer
Sang-Uk Choi1, Kyoung-Woo Kim1, Se Hoon Oh2, Jae Chan Kim1.
1
Ophthalmology, Chung-Ang University Hospital, Seoul, Korea
(the Republic of); 2Mechanical Engineering, Chung-Ang University,
Seoul, Korea (the Republic of).
A significantly decreased gene found in KC epithelium, Wnt10a,
was recently reported in an exome-wide associated study, to be
significantly associated with KC1. KC epithelium showed a strong
reduction in genes involved in the immune response and antivirus functional category, but upregulation of genes involved in
morphogenesis. This approach may help in understanding the
progressive degeneration of KC epithelium and the significance of
allergy/atopy in the pathogenesis of KC.
1. Cuellar-Partida G et al. Hum Mol Genet 2015; 24(17):5060-8.
The change in main outcomes at baseline and at one and three months
after botulinum toxin A injection. **p < 0.01, vs. baseline. Repeated
measures analysis of variance followed by Bonferroni post hoc were
used.
Commercial Relationships: Sang-Uk Choi, None; KyoungWoo Kim, None; Se Hoon Oh, None; Jae Chan Kim, None
RNA sequencing of the transcriptome of keratoconus and control
human corneal epithelium
Jingjing You1, Susan Corley2, Li Wen1, Chris Hodge3,
Michele C. Madigan4, 1, Gerard Sutton1, 3. 1Save Sight Institute,
University of Sydney, Sydney, NSW, Australia; 2NSW Systems
Biology Initiative, School of Biotechnology and Biomolecular
Sciences, University of New South Wales, Sydney, NSW, Australia;
3
Vision Eye Institute, Sydney, NSW, Australia; 4School of Optometry
and Vision Science, University of New South Wales, Sydney, NSW,
Australia.
Purpose: Keratoconus (KC), a degenerative corneal disease leading
to poor vision, involves both genetic and environmental factors,
although the gene pathways that contribute to the progression of the
disease are not established. In this study, we used RNA sequencing
to examine the transcriptome of corneal epithelium of KC and
control (myopia) patients and to investigate global expression of
differentially expressed genes.
Methods: Epithelial tissue removed from 5 KC and 4 control
(myopia) patients was used for RNA sequencing. Samples from
myopia patients undergoing PRK were used as controls. RNA was
extracted and prepared using a TruSeq Stranded Total RNA Library
Prep kit (Illumina), enriched using 12 cycles of PCR and sequenced
on NextSeq500 using a 75bp single read high output v2 run. The
generated data was analysed with Bioconductor packages, edgeR and
DESeq2.
The differentially expressed genes were subject to MetaCore analysis
for the discovery of genetic pathways/networks which may be
involved in KC. A validation cohort of a further 5 KC and 5 control
epithelial samples will be used to confirm the genes highly altered in
KC using qPCR.
Results: Differential gene expression analysis using edgeR and
DESeq2 showed a common set of 96 genes altered in KC compared
to control (Figure 1). The majority of these genes were downregulated in KC (77/96 genes), with 19 genes significantly upregulated in KC. Networks most significantly associated with the
down-regulated genes involved immune response and anti-virus
processes, while up-regulated genes were associated with tissue
morphogenesis.
Conclusions: RNA sequencing shows that KC epithelium has a
distinct gene profile compared to myopia (control) epithelium.
Figure 1: Both edgeR and DESeq2 have identified more than 100
differential expressed genes between KC and control epithelium, with
an intersection of 96 genes.
Commercial Relationships: Jingjing You, None; Susan Corley,
None; Li Wen, None; Chris Hodge, None; Michele C. Madigan,
None; Gerard Sutton, None
Support: Sydney Eye Hospital Foundation, Lions NSW Eye Bank,
The Ophthalmic Research Institute of Australia and Sydney Medical
School
Aldose reductase and oxidative stress markers in diabetic corneal
epithelium of enucleated human eyes. Margaret E. Young,
Thomas B. Redens, Donald E. Texada and Marlyn P. Langford
Department of Ophthalmology, Louisiana State University
Health Sciences Center, Shreveport, LA
Margaret Young. University Health, Shreveport, LA, Shreveport, LA.
Purpose: To document the distribution of aldose reductase (AR;
increased in hyperglycemic diabetic corneal epithelium) and markers
of oxidative stress and cell damage in corneal epithelium of diabetic
and non-diabetic enucleated human eyes.
Methods: Paraffin-embedded enucleated human eyes from 15
pathology enucleation cases (7 diabetic and 8 non-diabetic cases 9-65
years of age) were investigated. The expression of AR and oxidative
stress markers [xCT light chain of cystine/glutamate exchanger
(essential for transport of cysteine for maintaining antioxidant
potential through glutathione synthesis), oxidized DNA (8-OHdG;
oxidized 8-hydroxy-2'-deoxyguanosine marker of oxidative stress)
and annexin V (membrane damage)] was assessed in DAPI-stained
corneal epithelium by immunofluorescent antibody analysis.
Results: Immunoreactive AR was detected in basal columnar cells
of 4 of 7 (57%) diabetic corneas, but not in non-diabetic corneas.
AR was not detected in the wing cell layers, but was detected in
50% of all corneas with a flat surface epithelial cell layer. xCT was
predominantly expressed by flat surface epithelial cells, except in 2
corneas without a defined flattened surface epithelial layer. 8-OHdG
(oxidized nucleic acid) was expressed predominantly by basal
columnar corneal epithelial cells and weakly by wing and surface
epithelial cells. Annexin V was detected predominantly in the wing
and surface epithelial cells, but was detected in the columnar cells of
4 diabetic and 2 non-diabetic corneas.
Conclusions: The results present the distribution of AR expression
with markers of oxidative stress induced cell damage in corneas
of blind painful human eyes enucleated post trauma or diabetic
complication. AR was detected in diabetic columnar epithelial cells.
xCT and 8-OHdG were associated with annexin V expression in
the flattened surface epithelial cells. The results support increased
expression of AR by diabetic human corneal basal columnar
epithelium and surface epithelial cells.
Commercial Relationships: Margaret Young, None
Anti-neovascular effect of catechin on corneal alkaline burns in
rabbits
JaeWook Yang, Hye Sook Lee, Yoon Jin Lee, Tae Hoon Kang. Inje
Univ. Busan Paik Hospital, Busan, Korea (the Republic of).
Purpose: Alkali burns, which penetrate the cornea to a significant
degree, typically cause severe injury to corneal tissues. Alkali
burns can trigger inflammatory and immune-mediated pathways
that upregulate the expression of several angiogenesis factors. We
investigated the effect of a catechin on experimental corneal alkaline
burns in rabbits.
Methods: Corneal neovascularization (NV) was induced by applying
an 8-mm filter paper soaked in 1 N NaOH to the right central corneas
of rabbits for one minute. Seven days later, the rabbits were randomly
divided into three groups: the alkaline burn group (n=5, normal saline
instilled four times per day), and the 10 mg/mL catechin group (n=5,
10 mg/mL catechin instilled four times per day). The left eyes were
used as controls. On the 10th day after eyedrops, clinical outcomes
and histological changes of corneal structure were analyzed. Also
we investigated the effects of catechin on the expression of corneal
NV markers, identifying the mechanism of catechin suppression of
corneal NV.
Results: The alkaline burn produced significant NV (2.4±0.5) and
increased corneal thickness (961.4±17.36 μm). On day 10 after 10
mg/mL chatechin treatment, NV (1.4±0.5) and thickness (544.8±22.3
μm) of the cornea were markedly decreased in the catechin group
(p<0.05). In addition, the catechin improved the healing of the cornea
following alkaline burn, disrupting the corneal epithelial proliferation
and reducing the fibrotic changes of the stroma. The hallmarks
of angiogesis and inflammation including VEGF, CD31, MMP9,
macrophage, TNFα, ICAM-1, VCAM-1 and IL-1β were significantly
induced in the cornea by the alkaline burn, and these expression were
also suppressed by catechin. Furthermore, we demonstrated that
catechin suppressed alkali burn-induced corneal pathophysiological
changes by the NF-κB inacivation via blocking the Akt signaling
pathway.
Conclusions: In this study, we demonstrated that catechin was
markedly effective in healing alkali-burned corneas by modulating
the corneal opacity, NV, fibrosis and inflammation via blocking
the NF-κB. Therefore, catechin is possible promising material for
treatment of ocular surface disease related inflammation.
Commercial Relationships: JaeWook Yang, None; Hye Sook Lee,
None; Yoon Jin Lee, None; Tae Hoon Kang, None
Support: This study was supported by a grant from the Korea
Healthcare Technology R&D Project, Ministry of Health and Welfare
Affairs, Republic of Korea (grant #: HI12C0005)
Evaluation of Limbal and Central Corneal Epithelium Thickness
in Healthy Subjects using Anterior Segment Optic Coherence
Tomography
Tahir Kansu Bozkurt, Carolina Aravena Perez, Pichaya Chuephanich,
Dr. Chantaka Supiyaphun, Sophie X. Deng. Cornea Division, UCLA
Stein Eye Institute, Los Angeles, CA.
Purpose: To investigate limbal and corneal epithelial thickness and
investigate their correlation using Anterior Segment Optic Coherence
Tomography (OCT).
Methods: Twenty eyes from 10 healthy subjects were enrolled in this
study. The central corneal epithelial thickness map, linear scans of the
limbus at four locations, superior, inferior, nasal and temporal regions
were obtained using spectral-domain OCT (Optovue Inc., Fremont,
CA). Limbal epithelial thickness (LET) were measured manually
at the thickness area in each of the four regions of the limbus. The
corneal epithelium thickness (CET) map in the central (0-2 mm),
paracentral (2-5 mm) and peripheral (5-6mm)) areas were obtained
directly from the built-in analysis software of the OCT. Statistical
analysis was performed using SPSS program version 17 (SPSS Inc,
Chicago, Illinois, USA).
Results: The mean age of the normal subjects (7 female/3 male) was
35.2 ± 12.2 years. The mean CET was 53.5 ± 3.1 µm and LET in the
superior, inferior, nasal and temporal limbal region were 116.8 ± 19.7
µm, 106.9 ± 23.5 µm, 95.3 ± 14.6 and 91.1 ± 13.2 µm, respectively.
LET in the superior region was significantly thicker than that in
nasal and temporal regions (p < 0.01), but there was no significant
difference between LET in superior and inferior regions (p = 0.33).
There was a significant positive correlation between CET (6 mm) and
LET in the superior and inferior quadrants (p = 0.038 and p = 0.034,
respectively). LET in the superior limbus was significantly correlated
with CET in the superior, superonasal and superotemporal paracentral
areas (p < 0.05).
Conclusions: LET in the superior limbus is the thickest and
correlated with the central and superior paracentral CET in normal
subjects. AS-OCT could be utilized in the evaluation of cornea and
limbal epithelial thickness in normal and pathologic conditions.
Commercial Relationships: Tahir Kansu Bozkurt;
Carolina Aravena Perez, None; Pichaya Chuephanich, None; Dr.
Chantaka Supiyaphun, None; Sophie X. Deng, None
RNA-seq analysis of impact of PNN on gene expression and
alternative splicing in corneal epithelial cells
Stephen P. Sugrue1, Debra Akin1, Jeremy Newman2,
Lauren McIntyre2. 1Anatomy & Cell Biology, University of Florida,
Gainesville, FL; 2Molecular Genetics & Microbiology, University of
Florida, Gainesville, FL.
Purpose: The specialized corneal epithelium requires differentiation
specific for its role at the anterior surface of the eye, thus tight
maintenance of the differentiated qualities of the corneal epithelial
is essential. Pinin (PNN) is an exon junction component that has
dramatic implications on corneal epithelial cell differentiation and
may act as a stabilizer of the corneal epithelial cell phenotype.
Our studies revealed that PNN is involved in both transcriptional
repression complexes and spliceosomal complexes, placing PNN at
the fulcrum between chromatin and mRNA splicing. Transcriptome
analysis of PNN-knockdown cells revealed clear, reproducible
alterations in transcript profiles and splicing patterns of a subset of
genes that can significantly impact the epithelial cell phenotype.
Here, we further investigate PNN’s role in the regulation of gene
expression and alternative splicing (AS) in a corneal epithelial
context.
Methods: RNA-seq was used to determine differential gene
expression and AS events in authenticated human corneal epithelial
cells that carry a doxycycline-inducible PNN-knockdown shRNA
vector.
Results: Multiple genes and AS events were identified as
differentially expressed between PNN-knockdown and control
cells. Genes up-regulated by PNN-knockdown include a large
proportion that are associated with enhanced cell migration and ECM
remodeling such as MMPs, ADAMs, HAS2, LAMA3, CXCRs and
UNC5C. Genes down-regulated in response to PNN depletion include
IGFBP5, FGD3, FGFR2, PAX6, RARG and SOX10. AS events in
PNN knockdowns compared to controls were also more likely to
be detected and up-regulated. In particular, 60% of exon skipping
events, detected in only one condition, were detected in PNNknockdowns and of the shared exon skipping events, 92% of those
differentially expressed were more frequent in the PNN-knockdown.
Conclusions: These data suggest that lowering PNN levels in
epithelial cells results in dramatic transformation in the amount
and composition of splicing variants and that PNN plays a crucial
role in the selection of which RNA isoforms differentiating cells
produce. Many genes impacted by PNN-knockdown are known to
affect epithelial phenotype. This window into the complexity of
RNA splicing in the corneal epithelium implies that PNN exerts
broad influence over the regulation and maintenance of epithelial cell
phenotype.
Commercial Relationships: Stephen P. Sugrue, None; Debra Akin,
None; Jeremy Newman, None; Lauren McIntyre, None
Support: NIH Grant R01 EY007883, P30 EY021721
Influence of light emitting diode-derived blue light overexposure
on mouse ocular surface
Hyo Seok Lee1, Lian Cui1, 2, Ying Li1, 2, Ji Suk Choi1, Yeon Soo Kang1,
Won Choi1, In Cheon You3, Kyung Chul Yoon1. 1Department of
Ophthalmology, Chonnam National University Medical School and
Hospital, GWANGJU, Korea (the Republic of); 2Department of
Biomedical Science and Center for Creative Biomedical Scientists,
Chonnam National University, GWANGJU, Korea (the Republic
of); 3Department of Ophthalmology, Chonbuk National University
College of Medicine, Jeonju, Korea (the Republic of).
Purpose: To investigate the influence of overexposure to light
emitting diode (LED)-derived light with various wavelengths on
mouse ocular surface.
Methods: LEDs with various wavelengths were used to irradiate
6- to 8-week old female C57BL/6 mice at an energy dose of 50 J/
cm2, twice a day, for 10 consecutive days. The red, green, and blue
groups represented wavelengths of 630 nm, 525 nm, and 410 nm,
respectively. Untouched group (UT) was not exposed to LED light
and served as the untreated control. Tear volume, tear film break-up
time (TBUT), and corneal fluorescein staining scores were measured
on days 1, 3, 5, 7, and 10. Levels of interferon (IFN)-γ, interleukin
(IL)-1β, IL-6, and tumor necrosis factor (TNF)-α were measured
in the cornea and conjunctiva using a multiplex immunobead assay
at day 10. Levels of malondialdehyde (MDA) were measured
with an enzyme-linked immunosorbent assay. Flow cytometry,
2’7’-dichlorofluorescein diacetate (DCF-DA) assay, histologic
analysis, and terminal deoxynucleotidyl transferase-mediated dUTPnick end labeling (TUNEL) staining were also performed.
Results: TBUT of the blue group showed significant decreases at
days 7 and 10, compared with the UT and red groups (all p < 0.05).
Corneal fluorescein staining scores significantly increased in the blue
group when compared with UT, red, and green groups at days 5, 7,
and 10 (all p < 0.05). A significant increase in the corneal levels of
IL-1β and IL-6 was observed in the blue group, compared with the
other groups. The blue group showed significantly increased reactive
oxygen species production in the DCF-DA assay compared to the
UT, red and green groups (all p < 0.01) and increased inflammatory
T cells in the flow cytometry compared to the UT group (p = 0.023).
A significant increase in the density of TUNEL positive cells was
identified in the blue group compared with that in the other groups.
Conclusions: Overexposure to blue light with short wavelengths
(410 nm) can induce oxidative damage and apoptosis to the cornea,
which may manifest as increased ocular surface inflammation and
resultant dry eye
Commercial Relationships: Hyo Seok Lee, None; Lian Cui,
None; Ying Li, None; Ji Suk Choi, None; Yeon Soo Kang, None;
Won Choi, None; In Cheon You, None; Kyung Chul Yoon, None
TNF-R1 and FADD Mediate UVB-induced Activation of K+
Channels in Corneal Epithelial Cells
Peter M. Boersma1, Loren D. Haarsma2, Mark Schotanus1,
John L. Ubels1. 1Biology, Calvin College, Grand Rapids, MI;
2
Physics, Calvin College, Grand Rapids, MI.
Purpose: Exposure to ultraviolet B (UVB) radiation causes a K+
efflux from corneal epithelial cells due to activation of K+ channels.
This loss of intracellular K+ is an early step in UVB-induced
apoptosis, and inhibition of K+ efflux results in decreased rates of
apoptosis in corneal epithelial cells following UVB exposure (Ubels
et al. Exp Eye Res. 92:145; 145:26). Ligand-independent activation
of tumor necrosis factor receptor-1 (TNF-R1) by UVB results in
apoptosis (Sheikh et al. Oncogene 17:2555). This suggests that UVB
may activate K+ channels via TNF-R1. This study was designed to
investigate the roles of TNF-R1 and Fas-associated protein with death
domain (FADD), which is activated by TNF-R1, in the initiation of
the UVB-induced K+ efflux.
Methods: Ion chromatography was used to measure K+ loss from
human corneal limbal epithelial (HCLE) cells following exposure
to 150 mJ/cm2 UVB radiation or incubation with 50 ng/mL TNFα. Activation of K+ channels by 80 mJ/cm2 UVB was measured
by whole-cell voltage-clamp current recordings using standard
amphotericin-B perforated patch techniques. siRNA was used to
knock down TNF-R1 and FADD in HCLE cells.
Results: Exposure to UVB caused an increase in K+ channel currents
in less than 5 minutes. Knockdown of TNF-R1 resulted in a 50%
reduction in UVB-induced K+ current, while FADD knockdown
resulted in complete inhibition of UVB-induced K+ channel
activation. Cells exposed to UVB lost 45% of intracellular K+ within
20 minutes of exposure. After TNF-R1 knockdown UVB-induced K+
loss was eliminated, while FADD knockdown reduced UVB-induced
K+ loss to 15%. Cells incubated with TNF-α for 20 minutes exhibited
a 17% reduction of intracellular K+. These cells recovered normal
intracellular K+ levels by 45 minutes.
Conclusions: The data suggest that UVB directly activates TNF-R1,
which in turn may activate K+ channels via FADD. This conclusion
is supported by the fact that TNF-α also causes loss of intracellular
K+. This signaling pathway appears to be integral to UVB-induced K+
efflux, since knockdown of TNF-R1 or FADD inhibits UVB-induced
K+ efflux.
Commercial Relationships: Peter M. Boersma, None;
Loren D. Haarsma, None; Mark Schotanus, None; John L. Ubels,
None
Support: NIH grant R15 EY023836 and the Arnold & Mabel
Beckman Foundation Scholars Program
Preventive effects of green tea catechins on UV-induced
cytotoxicity in human corneal epithelium cells
AKIKO TOJU1, 2, Yuko Udaka1, Mayumi Tsuji1, Junichiro Kizaki1, 2,
Akiko Sasaki1, Katsuji Oguchi1. 1Phamacology, Showa University,
Tokyo, Japan; 2Ophthalmology, Showa University, Tokyo, Japan.
Purpose: The keratoconjunctiva of the ocular surface is directly
irradiated with solar ultraviolet light (UV) and exposed to many
stresses compared with organ tissue. Accordingly, various UVinduced diseases affecting keratoconjunctiva, such as cataracts
and photokeratitis have been based on epidemiological data. The
production of reactive oxygen species (ROS) and the mitogenactivated protein kinase (MAPK) signaling cascades have been
speculated as one of the mechanisms of UV-induced cytotoxicity.
Recently, it have reported that the tea catechin, especially,
(-)-Epigallocatechin Gallate (EGCG) and (-)-Epigallocatechin
3-(3”-O-Methyl) Gallate (EGCG 3"Me) have numerous bioactivity,
for example, anti-allergy, anti-oxidant, anti-inflammation, and
anticancer activity. Furthermore, recent study showed that treatment
with EGCG prevented the increment of apoptotic cells in dry eye
corneas. To elucidate the protective effects of catechin on the
UV-induced cytotoxicity in cultured human corneal epithelium
(HCE-T) cells, we determined utilizing we examined a role of MAPK
in protective effects of EGCG and EGCG 3"M on UV-induced
cytotoxicity in the HCE-T cells.
Methods: We cultivated HCE-T (the SV40-immnortalized
human corneal epithelial cell line; RCB No.2280) cells in
(DMEM:HamF12=1:1)+ 5%FBS+ 5mg/ml Insulin+ 10ng/ml Human
epidermal growth factor+ 0.5%DMSO+ 1%penicillin streptomycin in
5% CO2, 37 celsius environment. HCE-T irradiated UV (312nm, 4.94
mW/cm2, 296 mJ/cm2). HCE-T cells were treated with catechins for 1
hr before UV radiation. After UV exposure, the production of reactive
oxygen species (ROS), apoptotic rate and mitogen-activated protein
kinase (MAPK) activity were determined.
Results: The increment of ROS production, apoptotic cells, and
MAPK activity by UV exposure was prevented by treatment with
catechins. These EGCG-mediated cellular events were effectively
blocked by MAPK inhibitors.
Conclusions: These results suggest that EGCG might attenuate UVinduced cytotoxicity through the inhibition of the MAPK-signaling.
EGCG may be protective agent against UV-induced cytotoxicity in
HCE-T cells.
Commercial Relationships: AKIKO TOJU, None; Yuko Udaka;
Mayumi Tsuji, None; Junichiro Kizaki, None; Akiko Sasaki,
None; Katsuji Oguchi, None
The role of K+ and Ba2+ in inhibiting apoptosis in corneal
epithelial cells exposed to UVB
Courtney D. Glupker1, Peter M. Boersma1, John R. Leerar1,
Mark Schotanus1, Loren D. Haarsma2, John L. Ubels1. 1Biology,
Calvin College, Grand Rapids, MI; 2Physics, Calvin College, Grand
Rapids, MI.
Purpose: Exposure of corneal epithelial (HCLE) cells to ambient
UVB causes activation of K+ channels, loss of 50% of intracellular
K+ (K+i) within 10 min, activation of caspases and apoptosis.
Extracellular K+ at 25-100 mM inhibits these responses to UVB, and
K+i is restored by the Na/K pump within 90 min (Ubels et al. Exp
Eye Res. 92:145; 145:26). The latter is unexpected since the pump
is inhibited during apoptosis in lymphocytes. In the context of our
hypothesis that high [K+] in tears protects the corneal epithelium from
UVB by reducing loss of K+i, this study investigated effects of Ba2+
on UVB-induced K+ current and apoptosis of HCLE cells, effects of
UVB on Na/K ATPase activity and effects of [K+] on UVB-induced
caspase-3 activity.
Methods: HCLE cells were exposed to UVB at 80-150 mJ/cm2 and
incubated with or without Ba2+ for 4-6 hrs. The effect of Ba2+ on
UVB-induced K+ currents was measured by patch-clamp recording.
Caspase-activity assays and a TUNEL assay were used to determine
whether Ba2+ inhibits activation of UVB-induced apoptotic pathways.
Na/K ATPase activity was measured in cells exposed to UVB. Effect
of [K+] on UVB-induced caspase-3 activity was measured by addition
of 100 mM K+ to reaction medium.
Results: UVB-induced K+ currents were reduced in the presence of
5 mM Ba2+. UVB-exposed cells incubated with 1-5 mM Ba2+ showed
significant decreases in activation of caspases -9, -8, and -3 compared
to control cells not exposed to Ba2+. Apoptosis of cells exposed
to UVB, as measured by the TUNEL assay, was also inhibited in
the presence of 5 mM Ba2+. UVB had no effect on Na/K ATPase
activity. Addition of 100 mM K+ to the reaction medium significantly
decreased UVB-induced caspase-3 activity.
Conclusions: The data confirm that K+ current activation by UVB
leads to activation of the caspase cascade and apoptosis. Extracellular
Ba2+ inhibits this activation by preventing loss of intracellular K+,
inhibiting activation of downstream apoptotic pathways. The lack of
an effect of UVB on Na/K ATPase suggests that the pump is active
in recovery of intracellular K+, contributing to the protection of the
cornea from UVB. The data also suggest that UVB-induced caspase-3
activity requires low intracellular K+. This supports our hypothesis
that high extracellular K+ in tears protects the cornea from UVBinduced damage by limiting K+ efflux following UVB exposure.
Commercial Relationships: Courtney D. Glupker, None;
Peter M. Boersma, None; John R. Leerar, None; Mark Schotanus,
None; Loren D. Haarsma, None; John L. Ubels, None
Support: NIH Grant R15EY023836 and West Michigan Optometric
Scholarship
Analysis of ultraviolet-B irradiation induced changes in the
hTCEpi corneal epithelial cell line including effect of recovery
time on mRNA and protein expression
Kristin Olsson, Christopher Talbot, James K. Kubilus,
Thomas Linsenmayer. Integrative Physiology and Pathobiology, Tufts
University, Boston, MA.
Purpose: Corneal epithelial cells are constantly exposed to UV
irradiation. Previous studies focused on chicken corneal epithelial
cells and the molecule, ferritin, and its chaperone molecule, ferritoid,
which lend protection upon UV-B exposure. Current studies
transitioned to human telomerase-immortalized corneal epithelial
(hTCEpi) cells and focused on determining the normal apoptotic
response of these cells to UV-B irradiation. Future studies will seek
to elucidate protective capabilities of ferritin and ferritoid in hTCEpi
cells transfected with the molecules and exposed to UV-B.
Methods: hTCEpi cells were exposed to 12.5mJ/cm2 of UV-B
radiation based on previous studies. mRNA and protein isolated from
cells exposed to 12.5mJ/cm2 UV-B and from unexposed cells were
used in PCR arrays targeting human apoptotic markers and in western
blot analysis. Follow-up studies repeated these studies, but with
various recovery times (3, 7, 10, and 24 hours), to better determine
potential translational regulation.
Results: Initial PCR arrays showed changes in several apoptotic
markers, such as CD27, TNFRSF21, and NFkB1. Follow-up, targeted
qPCR analysis demonstrated results that mirrored initial PCR
array results with elevations in CD27 expression and decreases in
TNFRSF21 and NFkB1 levels. However, western blot against these
targets showed the opposite effect of UV-B irradiation for CD27
protein levels and reduced amounts of change in TNFRSF21 and
NFkB1 protein levels. Additional studies using different recovery
times after UV-B radiation exposure allowed for better determination
of the translational regulation that affects these pathways.
Conclusions: Based on previous studies and those described here,
we have shown that a dose of UV-B irradiation of 12.5mJ/cm2 causes
cell damage and alters mRNA levels of factors involved in apoptosis.
However, the protein levels of these genes do not always directly
correlate with the mRNA levels. To better determine the effect of
translational regulation of these cells following UV-B exposure,
various recovery times post-exposure were employed. Future studies
will determine if UV-B exposed hTCEpi cells transfected with
ferritin and ferritoid receive protection from these molecules or if
the apoptotic response demonstrated here in non-transfected cells
persists.
Commercial Relationships: Kristin Olsson, None;
Christopher Talbot, None; James K. Kubilus, None;
Thomas Linsenmayer, None
Support: NIH Grant R01 EY 013127
Identification of PNN-containing nuclear spliceosome and exon
junction complexes from HeLa and human corneal epithelial cell
Debra Akin, Stephen P. Sugrue. Anatomy & Cell Biology, University
of Florida, Gainesville, FL.
Purpose: The specialized corneal epithelium requires differentiated
properties, specific for its role at the anterior surface of the eye. Pinin
(PNN) has dramatic implications on corneal epithelial differentiation
and maintenance of the corneal epithelial cell phenotype. We have
shown that lowering of PNN levels in epithelial cells results in
dramatic transformation in the amount and composition of splicing
variants and that PNN plays a crucial role in the selection of which
RNA isoforms differentiating cells produce, implying that PNN
exerts broad influence over the regulation and maintenance of
epithelial cell phenotype through impacting RNA splicing. Here we
report the identification of a PNN complex that contains the essential
components for alternative splicing (AS).
Methods: HeLa and human corneal epithelial cells (HCET cells)
were engineered to express double-epitope-tagged versions of
PNN with bicistronic retroviral vectors pOZFH-C and pOZFH-N.
Subsequent to large-scale cell growth and nuclear extraction, the
PNN-containing nuclear complexes were isolated by tandem affinity.
MS/MS was then utilized for calculation of masses and peptide
identifications.
Results: We purified the PNN-containing complex(es) from HeLa
cells and analyzed components by mass spectrometry. Multiple core
spliceosomal components, such as SNRPD1, SNRPD3, splicing
factors, SR proteins, deadbox RNA helicases were found to be
complexed with PNN. Interestingly, PNN-complexes also contained a
high abundance of exon junction complex (EJC) components SAP18,
eIF4AIII, Magoh, Y14 and RNPS1, and Acinus. These data suggest
that PNN in associated with both the core spliceosome and the EJC.
PNN-containing complexes were then isolated from HCET cells and
Western blotting confirmed many of the proteins identified in Hela
cells.
Conclusions: These findings identify a molecular mechanism by
which PNN mediates modulation of RNA splicing and may influence
the epithelial transcriptome. It has been shown that the multiprotein
EJC is deposited on mRNAs upstream of exon–exon junctions and
serves as a key modulator of spliced mRNA metabolism. We suggest
that PNN may influence both RNA splicing and RNA stability
through its activity in the spliceosome and EJC.
Commercial Relationships: Debra Akin, None; Stephen P. Sugrue,
None
Support: NIH awards R01 EY07883, P30 EY021721
Effects of argon cold plasma treatment for ocular cells using
kINPen MED
Helena H. Reitberger1, Marta Czugala1, Anja K. Gruenert1,
Renate Schönebeck2, Friedrich E. Kruse1, Thomas A. Fuchsluger1.
1
Department of Ophthalmology, University Erlangen Nurnberg,
Erlangen, Germany; 2neoplas tools GmbH, Greiswald, Germany.
Purpose: The limited options available for the treatment of eye
infections and the increasing resistance of bacteria to antibiotics
emphasize the urge to find new therapeutic possibilities. The
disinfective potential of cold plasma therapy, due to the induction
of reactive oxygen and nitrogen species (ROS, RNS), makes this
approach a considerable possibility in the treatment of eye infections.
However, to ensure effective and safe application, optimal conditions
need to be found. In this study, we show the effects of cold plasma
treatment using kINPen MED (neoplas tools GmbH) on cultured
primary ocular cells.
Methods: The effect of cold plasma was tested on primary human
corneal limbal epithelial cells (pHCLEC) and human corneal
endothelial cells (HCEC-12). Cells were exposed to cold plasma
for 0.5, 1, 2, 5 and 10 min, with a distance of 5 cm and 90° angle
of incidence under constant moisturization. Metabolic activity as
measure for cell viability was analyzed by Cell Counting Kit - 8
(CCK-8) at 4 and 24 hours after treatment. Cell morphology and
density were examined by phase contrast microscopy. In addition,
the effects of cold plasma treatment on oxidative stress and apoptosis
induction were investigated by Western blot.
Results: Four hours after treatment 89.5% (0.5 min), 72.6% (1 min)
18.3% (2 min), 13.8% (5 min) and 15.6% (10 min) of the initial
metabolic activity could be measured in HCEC-12. Within 24 h, cells
treated for 0.5, 1 and 2 min have recovered to 131.4%, 112.7% and
64.4%, respectively. In these samples no changes in morphology or in
cell density could be noticed directly after treatment. After further 24
hours cell density visibly increased.
Western blot analysis did not show evident difference in GPX-1
levels between different exposure times as well as in comparison to
negative control, in both cell types. Slight PARP cleavage could be
detected in all treated HCEC-12 samples, while in pHCLEC this was
not the case.
Conclusions: In our study we could show that argon cold plasma
treatment with kINPen MED device for 0.5 – 2 min caused no effects
on the viability of ocular cells and does not trigger apoptosis or
induce oxidative stress in primary HCLEC. These findings support
the hypothesis, that in an optimized setting, high disinfective power
of cold plasma can be delivered while ensuring safety for the
surrounding ocular tissue.
Commercial Relationships: Helena H. Reitberger,
None; Marta Czugala, None; Anja K. Gruenert,
None; Renate Schönebeck; Friedrich E. Kruse, None;
Thomas A. Fuchsluger, None
The neurochemistry and morphology of functionally identified
corneal polymodal and cold-sensitive receptors
Abdulhakeem S. Alamri, James A. Brock, Jason J. Ivanusic. Anatomy
and Neuroscience, University of Melbourne, Melbourne, VIC,
Australia.
Purpose: Confocal microscopy studies have revealed that sensory
nerve terminations within the corneal epithelium can be distinguished
on the basis of their morphology and neurochemistry, but it has not
been confirmed that these different types of nerve terminal represent
distinct functional classes of sensory receptor. Here we extended
these studies by identifying the morphology and neurochemistry of
electrophysiologically defined corneal polymodal and cold-sensitive
receptors.
Methods: Eyes were isolated from guinea pigs that had been
euthanized by stunning and exsanguination. The eyes were mounted
in a recording chamber and superfused with physiological saline
(PS). Extracellular recordings from single polymodal or cold
receptors were made using glass pipette electrodes applied to the
corneal surface. The recording sites were marked by perfusing the
recording electrode with fluoro-gold (0.25% in PS). The corneas
were fixed and processed to reveal immunoreactivity for the transient
receptor potential channels TRPV1 or TPRM8.
Results: Polymodal receptors had either no ongoing impulse activity
or a low level of impulse activity (< 1 Hz), and were strongly
activated capsaicin (0.5 μM). At these recording sites (n=6), the
axons located immediately beneath the electrode were TRPV1immunoreactive (IR) and derived from an axon that ascended
from the sub-basal plexus to the superficial layer of the epithelium
where it branched into a number of fibres that each ran parallel to
the surface for a short distance before terminating in a small bulbar
ending (ramifying endings). Cold receptors had a continuous ongoing
rhythmic discharge of impulses at ~8 Hz that was increased by
cooling and inhibited by warming. At these recording sites (n=6),
the axons immediately beneath the electrode were TRPM8-IR
and originated from an axon that branched as it ascended from the
sub-basal plexus through the wing cell and squamous cell layers,
and terminated with large bulbar endings close to the surface of the
cornea (complex endings). Polymodal receptive endings (n=3) were
not TRPM8-IR and cold-sensitive endings (n=6) were not TRPV1-IR.
Conclusions: The findings confirm that in the most superficial layer
of the corneal epithelium that TRPV1 expressing nerve terminals are
polymodal receptors and TRPM8 expressing nerve terminals are cold
receptors.
Commercial Relationships: Abdulhakeem S. Alamri, None;
James A. Brock, None; Jason J. Ivanusic, None
The role of urokinase-type plasminogen activator receptor
(uPAR) in corneal epithelial wound healing process
Shoko Hiraki, Koji Sugioka, Aya Kodama, Yoshikazu Shimomura.
Ophthalmology, Kinki University, Osakasayama, Japan.
Purpose: urokinase-type plasminogen activator receptor (uPAR) is
glycosyl-phosphatidylinositol-anchored cell surface receptor which
is present in many tissues and has been identified in monocytes,
neutrophil granulocytes, endothelial cells, macrophages, fibroblasts,
and cancer cells. In this study, the role of urokinase-type plasminogen
activator receptor (uPAR) in corneal epithelial wound healing was
investigated.
Methods: The mice central corneal epithelium (3 mm in
diameter) was scraped using a Straight microsurgery blade under
a stereomicroscope. Expression of uPAR was determined by
immunohistochemistry. To investigate whether uPA and uPAR
expression were up-regulated by EGF treatments in human corneal
epithelial cells (HCEC), the protein and mRNA levels of uPA and
uPAR were determined. In addition, HCEC stably expressing u-PAR
was established. Using the uPAR overexpressing cells, in vitro
scratch assay was performed. The monolayer was mechanically
wounded using a sterile 10 μl pipette tip and 24 hours after
wounding, the lengths of scratched line were measured.
Results: uPAR were expressed in the leading edge upon the corneal
epithelial injury, and diminished after the wound was healed. In
vitro study showed that EGF-stimulated uPA and uPAR expression
increased in a time-dependent manner. In the results of scratch
assay, uPAR overexpressing HCEC increased the wound closure rate
compared with the control HCEC.
Conclusions: This study suggests that the uPAR expression probably
involve in migratory properties of corneal epithelial cells.
Commercial Relationships: Shoko Hiraki; Koji Sugioka, None;
Aya Kodama, None; Yoshikazu Shimomura, None
Femtosecond laser cutting of human corneas for subbasal nerve
plexus evaluation
Rudolf F. Guthoff1, Heiko Richter2, Andreas Wree3, Carl F. Marfurt4,
Bhavani S. Kowtharapu5. 1Institute for Biomedical Engineering,
Rostock University Medical Center, Rostock, Germany; 2LLS
Rowiak LaserLabSolutions GmbH, Hannover, Germany; 3Institute
of Anatomy, Rostock University Medical Center, Rostock, Germany;
4
Indiana University School of Medicine-Northwest, Gary, IN;
5
Department of Ophthalmology, Rostock University Medical Center,
Rostock, Germany.
Purpose: Tissue sectioning is an essential but time consuming part of
histology which demands special expertise and man power. The aim
of the present work is to establish a rapid, reliable and reproducible
sectioning method that is applicable to all kinds of tissues, using a
femtosecond laser based microtome. This technique is exemplified in
human corneas for standardized visualization of the subbasal nerve
plexus (SBP).
Methods: Trephined healthy human corneal buttons were fixed
and processed using a commercially available femtosecond laser
based microtome to obtain surface parallel tissue sections of the
anterior stroma. Positioning and control of section plane by OCT
(optical coherence tomography) distinguished corneal epithelium
from other layers of the cornea during the cutting process. A near
infrared femtosecond laser (wavelength 1030 nm; pulse duration
300 femtoseconds; output power between 2.5 W – 6.0 W; repetition
rate 10 MHz) was focused onto the cornea approximately 70-90
µm from the anterior side to induce material separation. Corneal
sections containing the entire epithelium (50-60 µm) and part of
the anterior stroma (20-30 µm) were stained following standard
immunohistochemical procedures with anti-neuronal β-III-tubulin
antibody for visualization of the corneal nerves and compared with
similarly stained anterior corneal sections (100-140 µm) obtained by
a cryo-microtome.
Results: SBP staining pattern of femtosecond laser microtome
prepared anterior stromal sections show resemblance to cryomicrotome obtained corneal sections. Laser cut corneal sections
also yielded good visualization of SBP as the interference from the
stromal nerves was largely minimized.
Conclusions: Tissue sectioning with the use of femtosecond laser is
completed within approximately 15 minutes and offers an easier way
than the currently available methods. Furthermore, without any tissue
pre-processing steps involved, this method is faster than regular
sectioning methods. This methodology can also be adopted easily
for cutting all kinds of biological samples, including hard tissue
elements, as it offers a more standardized way of tissue sectioning.
Commercial Relationships: Rudolf F. Guthoff, None;
Heiko Richter, None; Andreas Wree, None; Carl F. Marfurt,
None; Bhavani S. Kowtharapu, None
Molecular Mechanism of Ocular Surface Damage: Applications
to Dry Eye and Wound Healing Models on In Vitro
Reconstructed Human Corneal Tissues
Yulia Kaluzhny, Miriam W. Kinuthia, Alena Plotkin, Patrick Hayden,
Mitchell Klausner. MatTek Corporation, Ashland, MA.
Purpose: Current methods used to investigate mechanisms of
corneal wound healing (CWH) and pathogenesis of dry eye disease
(DED) utilize monolayer cell cultures or animals. However these
methods present numerous disadvantages and there is a need for more
physiologically relevant, human-based in vitro models for CWH and
DED research.
Methods: This study utilized EpiCorneal tissue model comprised
of normal human corneal epithelial cells that are cultured at the
air-liquid interface. Corneal wounds were introduced by abrasion
or chemicals (1N NaOH). Wounded tissues were cultured in the
presence or absence of human corneal keratocytes (HCK) or EGFR
inhibitor (erlotinib, 10 μM). A DED model was generated by placing
EpiCorneal tissues under desiccating stress conditions (40% RH,
40°C, and 5% CO2) that stimulate morphological, cellular, and
molecular changes relevant to dry eye.
Results: CWH was analyzed by transepithelial electrical resistance
(TEER), histology, confocal microscopy, and gene expression. TEER
recovered to 933.7/502.4 Ω*cm2 in the presence/or absence of HCK
in 4 days post-wounded cultures. mRNA expression was analyzed
using a 96-gene wound healing microarray. 13 genes (including
collagen, integrin, chemokine, and protein kinase families) were
up-regulated in the EpiCorneal tissues 24h post-abrasion in the
absence of HCK and 16 genes (including WNT, FGF, small GTPases,
chemokine, and integrin families) were up-regulated in the presence
of HCK, but not in control cultures. DED was analyzed by TEER,
histology, tissue viability, mucins and tight junction (TJ) protein
expression. Dramatic reduction in tissue thickness was observed after
48h in DSC that coincided with decreased expression of mucins,
increased TEER and atypical expression of TJ proteins. Topical
application (25 μl/tissue) of lubricant gel drops (GenTeal, Alcon)
improved tissue morphology and barrier function.
Conclusions: The results demonstrate that the in vitro organotypic
human corneal tissue structurally and functionally reproduces CWH
and DED. The model will avoid species extrapolation, be more
cost effective and more reproducible than animal methods, and will
facilitate drug discovery by allowing screening and optimization of
active pharmaceuticals prior to clinical studies.
Commercial Relationships: Yulia Kaluzhny, MatTek;
Miriam W. Kinuthia, MatTek; Alena Plotkin, MatTek;
Patrick Hayden, MatTek; Mitchell Klausner, MatTek
The Effect of Notch Signaling Pathway on the Proliferation and
Differentiation of Corneal Epithelial Cells
Wei Li1, 2, Tingting Liu1, Yangluowa Qu1, Hui Lin1, Changkai Jia1, 2,
Zuguo Liu1, 2. 1Dept. Ophthalmology & Visual Science, Eye Institute
of Xiamen University, Xiamen, China; 2Xiamen University affiliated
Xiamen Eye Center, Xiamen, China.
Purpose: To investigate the expression of Notch signaling pathway
in ocular surface epithelial cells, and to determine the effect of Notch
signaling pathway on the proliferation and differentiation of the
ocular surface epithelial cells.
Methods: Normal human limbal tissues were collected and
embedded in OCT for cryosection. Limbal epithelial cells were clonal
cultured in SHEM with 3T3 feeder layers. TKE2 cells were cultured
in KSFM or with additional calcium or 10% FBS. Morphological
change of TKE2 cells was observed and photographed. The
expression of proliferation markers and Notch related factors in
TKE2 cells, human limbal tissue and epithelial colonies were
performed by immunohistochemistry, fluorescence and Western blot.
Results: Notch receptors Notch1, Notch2, and Notch4, but not
Notch3 were expressed in human limbal and corneal epithelium, as
well as limbal epithelial colonies. Notch ligands such as Jagged1
and Jagged2 were expressed in limbal corneal epithelium. The down
stream gene of Notch signal Hes1 were expressed in central corneal
epithelium, and co-localized with stem cell marker p63 in corneal
epithelial colonies. TKE2 cells cultured in KSFM showed strong
expression of p63, PCNA, Ki67, K14, and K16. Notch1 and Hes1
were expressed in the nuclei, while Notch2 and Hey1 were expressed
in the cytoplasm and nuclei of TKE2 cells in KSFM medium.
However, the expression of p63, Notch1, Notch2, Jagged1, Jagged2
and Hes1 were significantly reduced, while Math1, as the Hes1
inhibitor was highly increased in TKE2 cultured in high calcium or
serum containing medium.
Conclusions: Notch signaling pathway is activated in human limbal
epithelial tissues, human limbal epithelial clones and TKE2 cell
line with high proliferative capacity, but not in TKE2 cell lines in
differentiated state. Notch signaling pathway may play an important
role in maintaining proliferative potential and phenotype of limbal
stem cells.
Commercial Relationships: Wei Li; Tingting Liu, None;
Yangluowa Qu, None; Hui Lin, None; Changkai Jia, None;
Zuguo Liu, None
Support: This work was supported in part by grants from the
Chinese National Key Scientific Research Project (2013CB967003
[to WL]), the National Natural Science Foundation of China (NSFC,
No. 81270977 [to WL], No. 81470602 [to WL], No. 81270978 [to
ZL], No. 81330022 [to ZL]), Chinese National Health and Family
Planning Commission Project (WKJ-FJ-26 [to WL]), Fundamental
Research Funds for the Central Universities of China (No.
20720150171 [to WL]).
Rock inhibitor facilitates mouse and rat corneal epithelial cells
culture
Changkai Jia, Sanming Li, Yi Liao, Juan Li, Liying Zhang, Wei Li,
Zuguo Liu. Medical College of Xiamen University, Eye Institute of
Xiamen University, Xiamen, China.
Purpose: In vitro primary culture of mouse or rat corneal epithelial
cells (CECs) remains as a big obstacle to be resolved, which largely
hinders our understanding about the molecular mechanisms to
maintain the phenotypes as well as physiological functions of corneal
epithelial cells. Therefore, establishment of a feasible in vitro culture
protocol for animal originated epithelial cells is urgently required.
Methods: Freshly harvested mouse or rat corneas were digested in
culture medium with 2U dispase ‖overnight. Then the epithelial layer
was detached from stroma, and further digested into single cells with
0.05% Trypsin-EDTA. Afterwards, single cells were plated onto the
collagen IV pretreated dish, and cultured with defined keratinocyte
serum free medium (D-KSFM) or supplemental hormonal epithelial
medium (SHEM) with the addition of Rock inhibitor Y27632,
respectively. For cultures with serum supplement, NIH-3T3 cells
were used as feeder layers. When cells reached confluency, some
cells were used for further passages, while the others were harvested
for qRT-PCR, Immunocytochemistry and western blot to examine
their phenotypical markers.
Results: Using both D-KSFM and SHEM media supplemented with
Y27632, rat corneal epithelium could be easily cultured for up to
10 passages. In comparison, the culture of mouse corneal epithelial
cells required prolonged culture time. While mouse CECs could be
cultured by SHEM medium for a few generations, cells maintained
D-KSFM with Y27632 propagated continuously. CEC specific
markers Pax6, K12, P63 and K14 were examined by qRT-PCR,
Immunocytochemistry and Western blot. Although these markers
were positive in primary culture, K12 was sharply decreased after
passage. Moreover, Pax6, a transcription factor essential for CECs
phenotype maintenance, was gradually decreased. Serving as the
corneal epithelial progenitor markers, p63 was maintained at a
high level and K14 expression was increased during culture. Skin
epithelium marker K10 was negative for all cultured cells.
Conclusions: Rock inhibitor is an essential supplement to promote
mouse or rat CECs adherence and proliferation in vitro, which
facilitates mouse and rat corneal epithelial cells culture, while the
underline mechanism needs further investigation.
Commercial Relationships: Changkai Jia, None; Sanming Li,
None; Yi Liao, None; Juan Li, None; Liying Zhang, None; Wei Li,
None; Zuguo Liu, None
Support: Chinese National Key Scientific Research Project
2013CB967003
Corneal changes with Latanoprost use
Yasser H. Mohamed1, 2, Masafumi Uematsu1. 1Ophthalmology,
Nagasaki University, Nagasaki, Japan; 2Ophthalmology, El-Minia
University, El-MInia, Egypt.
Purpose: To determine the corneal changes after usage of
Latanoprost along different periods of time.
Methods: Male white Japanese rabbits (KBT Oriental, Tosu, Japan)
were used in this study. We divided the animals into 6 groups:
Three groups for Latanoprost exposure corneas (immediately after
latanoprost exposure, after 24 hour of exposure, after one week of
exposure) and three groups as control groups with Hank’s Balanced
Salt Solution (HBSS) exposure. Corneal transepithelial electrical
resistance (TER) and transmission electron microscopy (TEM), and
scanning electron microscopy (SEM) examinations were done for all
groups.
Results: There was a significant decrease in the corneal TER after
immediate exposure of the cornea to latanoprost and no significant
changes in the corneal TER after exposure of the cornea to other
two groups of latanoprost or control groups. In the first group of
immediate Latanoprost exposure, some of the superficial cells of
the corneal epithelium were damaged, exhibited lost or degenerated
microvilli, lost their adhesions with adjacent cells, and cracks
appeared between cells by SEM examination. Separation between
epithelial cells was confirmed with TEM examination. The superficial
cells of the corneas of the second and third groups of Latanoprost
exposure and control groups were normal in appearance with normal
microvilli and normal adhesions between cells.
Conclusions: Latanoprost has acute transient toxic effect on the
epithelial cells of the cornea. The regenerative power of the corneal
epithelium counteracts of this toxic effect within 24 hours.
Commercial Relationships: Yasser H. Mohamed, None;
Masafumi Uematsu, None
Toxicity of glaucoma drugs on corneal epithelial cells
Alexandra Robciuc1, 2, Suvi Ruokonen3, Joanna Witos3,
Antti H. Rantamaki3, Susanne Wiedmer3, Juha M. Holopainen1.
1
Helsinki Eye Lab, University of Helsinki, Helsinki, Finland;
2
Genomics and Biomarkers, National Institute for Health and
Welfare, Helsinki, Finland; 3Department of Chemistry, University of
Helsinki, Helsinki, Finland.
Purpose: Glaucoma drugs (GDs) are intended to reduce elevated
intraocular pressure and prevent damage to the optic nerve. They
are applied topically and increase aqueous humor outflow or reduce
aqueous humor production. Majority of the GDs are hydrophobic,
similarly to benzalkonium chloride (BAK), and therefore may disturb
the organization of the cellular membranes. The aim of our study was
to determine the toxicity of the GDs on the ocular surface cells.
Methods: The toxicity of clinically efficient doses of latanoprost,
timolol maleate, brimonidine tartrate, brinzolamide and pilocarpine
hydrochloride was measured at 4, 8, 16 and 24h, using a cell culture
of human corneal epithelial cells. The drugs interaction with the
plasma membrane was analyzed using the hemolysis assay and
capillary electrophoresis. The capacity of the drugs to induce
endoplasmic reticulum (ER) stress response was investigated using
immunoblotting.
Results: The toxicity assay showed that all GDs affect the viability
of the epithelial cells to variable degrees. Early toxicity was
measured for pilocarpine 4% and brimonidine 0.1% and 0.15%
with 60% cell death at 4h, while pilocarpine 2% and latanoprost
0.005% showed almost 100% toxicity but only after 16h. Timolol
0.5%, as well as pilocarpine 1%, induced 40% cell death at 24h.
However, the hemolysis assay was negative for all GDs and capillary
electrophoresis confirmed that the GDs interact with the lipid
membrane but the interaction is weak and cannot account for the cell
death through lysis. Immunoblotting for ER stress markers revealed
that the drugs activate the ER stress pathways but only timolol,
latanoprost and, to a lower extent, brimonidine have the capacity to
induce apoptosis through upregulation of CHOP.
Conclusions: Our study suggests that all GDs affect the viability
of the corneal epithelial cells and that the toxicity is determined by
their interaction with intracellular components. Topical application
implies that GDs need to penetrate the cornea in order to reach their
specific targets, consequently, their toxicity could explain, at least in
part, their local side effects. These findings support the conclusions
from the PESO study, which showed that filtering surgery can be
considered as first line of treatment and furthermore suggest that
intraocular long-retention implants could be safer to the ocular
surface compared to the topical application of anti-glaucoma drugs.
Commercial Relationships: Alexandra Robciuc, None;
Suvi Ruokonen, None; Joanna Witos, None; Antti H. Rantamaki,
None; Susanne Wiedmer, None; Juha M. Holopainen, CromaPharma (S)
Support: Sigrid Juselius Foundation, Helsinki University Central
Hospital Research Foundation and the Finnish Eye Foundation grants
Ophthalmic Manifestations of X-Linked Reticulate Pigmentary
Disorder
Ahmed F. Omar1, 2, Vinod V. Mootha1, Stefano Pensiero3.
1
Ophthalmology, UT Southwestern Medical Center, Dallas, TX;
2
Ophthalmology, Faculty of Medicine - Assiut University, Assiut,
Egypt; 3IRCCS Burlo Garofolo, Children’s Hospital of Trieste,
Trieste, Italy.
Purpose: X-linked Reticulate Pigmentary Disorder (XLRPD) is
a rare genetic disorder described in only a few families to date.
Affected males have diffuse reticulate pigmentation of skin, corneal
dyskeratosis as well as other systemic manifestations. We report the
ocular manifestations of 6 cases of XLRPD.
Methods: Chart review of 6 male subjects from 4 families with
XLRPD.
Results: The age range was 7-35 years. All subjects presented
shortly after birth with repeated lung and urogenital infections and
show classic skin and hair findings. Three subjects have immediate
female family members with cutaneous manifestations. The average
age at presentation of the ocular findings was 2 years. The visual
acuity ranged from 20/20 to hand motion. All subjects had severe
photophobia at presentation. Corneal examination in all subjects
showed bilateral raised, nodular anterior stromal lesions, pannus, and
anterior stromal vessels. Tear production testing with local anesthesia
showed an average (n=4) of 33mm wetting of the Schirmer strip
paper. Fluorescein staining showed whorl epithelopathy with
an 0.5 mm epithelial defect in one eye of one subject, bilateral
punctate staining compatible with dryness in another subject, and
negative staining over the corneal nodules in two other subjects.
The remainder of anterior segment examination was unremarkable
in all subjects. Fundus examination of three subjects under general
anesthesia revealed bilateral diffuse retinal pigmentation and pale
optic nerve in one subject and was unremarkable in the other 2
subjects. Electrophysiological studies of the subject with retinal and
optic nerve changes revealed bilateral delayed visual evoked response
and unilateral mild reduction of scotopic electroretinogram response.
Five eyes of 3 subjects underwent surgical procedures such as
phototherapuetic keratectomy (PTK) and lamellar keratoplasty (LK).
Recurrence of the nodular anterior stromal lesions and haze was
noted after the surgical procedures in all 5 eyes.
Conclusions: XLRPD results in significant visual disability
secondary to severe photophobia and corneal scarring. The corneal
lesions and scarring may recur after PTK or LK.
Commercial Relationships: Ahmed F. Omar, None;
Vinod V. Mootha; Stefano Pensiero, None

Session 412 Corneal Epithelium

Transcription

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