ARVO 2015 Annual Meeting Abstracts 164 Cornea endothelium

Transcription

ARVO 2015 Annual Meeting Abstracts
164 Cornea endothelium
Sunday, May 03, 2015 3:15 PM–5:00 PM
Exhibit Hall Poster Session
Program #/Board # Range: 1141–1183/D0043–D0085
Organizing Section: Cornea
Program Number: 1141 Poster Board Number: D0043
Presentation Time: 3:15 PM–5:00 PM
Effect of dexamethasone on the phenotype of cultured corneal
endothelial cells
Veronique Beaulieu Leclerc1, 2, Olivier Roy1, 2, Stephanie Proulx1,
2 1
. Axe médecine regénératrice, Centre de recherche du Centre
hospitalier universitaire (CHU) de Québec, Québec, QC, Canada;
2
Ophtalmologie, Université Laval, Quebec, QC, Canada.
Purpose: Human corneal endothelial cells (HCEC) tend to lose
their endothelial morphology in culture, a phenomenon known as
endothelial-to-mesenchymal transition (EndMT). The purpose of
this study was to evaluate the ability of a synthetic glucorticoïd,
dexamethasone (Dex), to block EndMT of cultured HCEC.
Methods: HCEC (n=4 populations, passage 2) with a mixed
phenotype were passaged and grown in a proliferation medium
containing Dex (1 mM). The medium was changed for a maturation
medium still containing Dex after reaching confluency and the cells
were cultured for another 7 days. Two other HCEC populations were
cultured in the proliferation medium alone until confluency and Dex
was added to the maturation medium for 7 days. Cultures without
Dex were used as controls. Morphology was assessed by phase
contrast microscopy. Immunofluorescence and western blots were
performed (endothelial functionality markers ZO-1, Na+/K+ATPase
and pan-cadherin; mesenchymal markers α-SMA and type I collagen)
in order to analyze the phenotype.
Results: Adding Dex during the proliferation phase of HCEC had
no significant effect on the morphology of the cells. However,
morphology was improved during the maturation phase.
Immunofluorescence and western blot analysis showed that when
Dex was used in both cultivation phases, it reduced the expression of
mesenchymal markers, compared to the controls. Dex also improved
the localisation of ZO-1 at cell junctions and increased Na+/K+ATPase
expression. When Dex was only used in the maturation phase, the
morphology greatly improved, from mixed/fibroblastic to endothelial.
Expression of type I collagen was not significantly different (p>0.05)
and expression of α-SMA was higher than the controls. Cells that
went through the maturation phase in the presence of Dex had
an increased expression of the endothelial markers ZO-1, Na+/
K+ATPase and pan-cadherin as well as a better cytolocalisation when
compared to the controls (without Dex) and compared to cells in the
proliferation phase.
Conclusions: Addition of Dex to the culture media, in both culture
phases, helps to acquire an endothelial phenotype. Results also
demonstrate that the maturation of HCEC in the right conditions
plays a role in the establishment of an endothelial phenotype and
expression of function-related proteins.
Commercial Relationships: Veronique Beaulieu Leclerc, None;
Olivier Roy, None; Stephanie Proulx, None
Support: NSERC, RRSV, ThéCell, Wilbrod-Bhérer studentship of
Laval University.
Nuclear YAP-promoted proliferation can be induced by
lysophosphatidic acid via the PI3K and ROCK pathways in
contact-inhibited human corneal endothelial cells
Yi-Jen Hsueh1, Hung-Chi Chen1, Sung-En Wu2, Tze-Kai Wang1, JanKan Chen2, David H. Ma1. 1Stem Cell Laboratory of Ophthalmology,
Chang Gung Memorial Hospital, Taoyuan, Taiwan; 2Department of
Physiology, College of Medicine, Chang Gung University, Taoyuan,
Taiwan.
Purpose: Silence of p120-catenin has shown promise in inducing
proliferation in human corneal endothelial cells (HCECs), but
there is concern regarding gene recombination in potential clinical
applications. We aimed to develop ex vivo expansion of HCECs
using natural compounds, and we hypothesized that lysophosphatidic
acid (LPA) can unlock the mitotic block in contact-inhibited HCECs
via enhancing nuclear translocation of yes-associated protein (YAP).
Methods: Collagenase-isolated HCEC aggregates from stripped
Descemet’s membrane were cultured to 14 days, and treated by 100
nM control or YAP-1 siRNA. Cells were further treated with 20
μM LPA and 10 μM BrdU . Likewise, post-confluent b4g12 cells
(HCEC cell line) cultured to 4 days under the similar condition.
Immunofluorescent staining was performed to demonstrate YAP1, ZO-1, Na/K-ATPase, SMA and BrdU labeling. B4g12 cells
was pretreated with LY294002 (PI3K inhibitor, 20 μM), PD98059
(ERK1/2 inhibitor, 20 μM), SB203580 (p38 inhibitor, 20 μM) or
Y27632 (ROCK inhibitor, 20 μM) for pathway assay. Western blot
analysis was applied to evidence the regulation of proliferation by
targeting p27/p21 and cyclin D1.
Results: Firstly, we verified that exogenous YAP could induce
cell proliferation in contact-inhibited HCEC monolayers and
post-confluent B4G12 cells. In B4G12 cells, enhanced cyclin D1
expression, reduced p27KIP1/p21CIP1 levels and the G1/S transition
were detected upon transfection with YAP. Secondly, we confirmed
that LPA induced nuclear expression of YAP and promoted cell
proliferation. Moreover, PI3K and ROCK, but not ERK or p38, were
required for LPA-induced YAP nuclear translocation. Finally, cells
treated with LPA or transfected with YAP remained hexagonal in
shape, in addition to unchanged expression of ZO-1, Na/K-ATPase,
and SMA, suggestive of a preserved phenotype, without endothelialmesenchymal transition (EnMT).
Conclusions: Our findings indicate an innovative strategy for ex vivo
cultivation of HCECs for transplantation and cell therapy.
Commercial Relationships: Yi-Jen Hsueh, None; Hung-Chi Chen,
None; Sung-En Wu, None; Tze-Kai Wang, None; Jan-Kan Chen,
None; David H. Ma, None
Support: CGMH CMRPG3C0521-3
Clinical Trial: 103-0826C
Activation of RhoA-ROCK-BMP-miR302 Network Reprograms
Human Corneal Endothelial Cells to Neural Crest Progenitors
Yingting Zhu, Xin Liu, Wenjuan Lu, Szu-Yu Chen, Scheffer C. Tseng.
Tissue Tech, Inc, Miami, FL.
Purpose: To explore the mechanism of reprogramming human
corneal endothelial cell (HCEC) monolayers into neural crest (NC)
progenitors by knockdown with p120-catenin (p120)-Kaiso siRNAs.
Methods: HCEC monolayers derived from stripped Descemet
membrane were cultured to 7 days and treated with 100 nM of p120
and Kaiso siRNA in MESCM for up to 5 weeks. Before termination,
cells were labeled with 10 μM BrdU for 4 hours. Transcript
expression and cytolocalization of NC, embryonic stem cell (ESC),
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
keratocyte, and cell cycle markers, miR 302s, and signaling markers
was determined by RT-qPCR and immunostaining. Western blotting
was used to measure respective proteins.
Results: As reported previously, MESCM promoted expansion
the size of HCEC monolayers to 4.4 ± 0.6 mm without but to 11.0
± 0.6 mm in diameter with weekly knockdown by p120-Kaiso
siRNAs from 1/8 corneoscleral rim after 6 weeks of culture. The
use of MESCM medium containing LIF was important because
LIF promoted expansion of HCEC monolayers by delaying contact
inhibition via activation of LIF-JAK-STAT3 signaling and through
downregulating senescence-related p16 while upregulating cell
cycle-promoting genes. Addition of p120-Kaiso siRNA after 1-2
weeks of culture could further promote expansion by reprogramming
HCECs into their progenitors through activation of RhoA-ROCKcanonical BMP signaling. Further analysis indicated that activation of
canonical BMP was required for reprogramming because knockdown
of BMP receptors abolished such reprogramming, but alone was not
sufficient to lead to such reprogramming since addition of BMP4/6
(25 and 50 ng/ml, respectively) activated canonical BMP signaling,
promoted expression of ESC and NC markers but did not lead to
nuclear translocation of Oct4-Sox2-Nanog complex. Canonical BMP
signaling required RhoA-ROCK signaling triggered by knockdown
with p120-Kaiso siRNAs to activate miR 302b/c-Oct4-Sox2-Nanog
network for reprogramming. Finally, the NC progenitor status was
confirmed by differentiation of HCEC progenitors into keratocanexpressing keratocytes.
Conclusions: This new strategy of reprogramming of HCECs
into NC progenitors by activation of RhoA-ROCK-BMP-miR 302
Network could be used to engineer HCEC surgical grafts containing
HCECs for endothelial keratoplasties to meet a global corneal
shortage in treating corneal blindness caused by corneal endothelial
dysfunction.
Commercial Relationships: Yingting Zhu, Tissue Tech, Inc (E),
Tissue Tech, Inc (F), Tissue Tech, Inc (P); Xin Liu, None; Wenjuan
Lu, None; Szu-Yu Chen, Tissue Tech, Inc (E), Tissue Tech, Inc (F);
Scheffer C. Tseng, Tissue Tech, Inc (E), Tissue Tech, Inc (F), Tissue
Tech, Inc (I), Tissue Tech, Inc (P)
Support: EY022502
The different binding properties of cultured human corneal
endothelial cell subpopulations to Descemet’s membrane
components
Munetoyo Toda1, Morio Ueno1, Asako Hiraga1, Kazuko Asada1,
Takahiro Nakamura1, Michio Hagiya1, Naoki Okumura2, Noriko
Koizumi2, Junji Hamuro1, Shigeru Kinoshita1. 1Department of
Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto,
Japan; 2Department of Biomedical Engineering, Faculty of Life and
Medical Sciences, Doshisha University, Kyotanabe, Japan.
Purpose: Human corneal endothelial cells (HCECs) have poor
proliferative ability under in vitro culture conditions. The tendency to
enter into cell senescence or phase transition (epithelial-mesenchymal
transition (EMT), cell senescence, and fibrosis) during cultivation
produces different subpopulations from HCECs. To date, we have
developed definitive subpopulations based on their cell surface
markers. To clarify the adherent properties of these subpopulations,
we compared the binding ability of cultured HCEC subpopulations
to major Descemet’s membrane components that distribute to the
endothelial face; i.e., laminin-511, -411, Type IV collagen, and
proteoglycans.
Methods: Each subpopulation was prepared by controlling the
culture conditions or by using magnetic cell separation, and then
confirmed by staining with several cell surface markers. To examine
the binding abilities of HCEC subpopulations, the cells were added
to 96-well culture plates immobilized with collagens, laminins, or
proteoglycans, and the plates were then centrifuged. The attached
cells were then evaluated under a phase contrast microscope.
Results: Both the fully differentiated mature HCEC subpopulation
and the EMT-phenotype subpopulation were found to be attached
to laminin or collagen coated plates. Among the examined
laminins, HCEC subpopulations were most strongly bound to
laminin-511 and weakly bound to Perlecan, Agrin, and TSP-1.
Interestingly, the binding properties to laminins were different
among these subpopulations. Although the level of attached cells
to the laminin-411 coated plate was the same among the HCEC
subpopulations, the fully differentiated mature HCEC subpopulation
was significantly more tightly bound to laminin-511 than was the
EMT-phenotype subpopulation.
Conclusions: Our results suggest that the binding ability of HCECs
to the major Descemet’s membrane components is distinct among
subpopulations of cultivated HCECs. Moreover, the simple methods
used in this study are effective for evaluating the interaction between
HCECs and extracellular matrix components.
Commercial Relationships: Munetoyo Toda, None; Morio Ueno,
Santen Pharmaceutical Co. (P), Senju Pharmaceutical Co. (P); Asako
Hiraga, None; Kazuko Asada, None; Takahiro Nakamura, None;
Michio Hagiya, JCR Pharmaceuticals Co. (E); Naoki Okumura,
JCR Pharmaceuticals Co. (P), Senju Pharmaceutical Co. (P); Noriko
Koizumi, Senju Pharmaceutical Co. (P), JCR Pharmaceuticals
Co. (P); Junji Hamuro, None; Shigeru Kinoshita, Otsuka
Pharmaceutical Co. (C), Santen Pharmaceutical Co. (P), Senju
Pharmaceutical Co. (P)
The feasibility of recombinant human laminin-511 E8 fragments
for human corneal endothelial cell cultivation
Kazuya Kakutani1, Naoki Okumura1, 2, Ursula Schlotzer-Schrehardt3,
Friedrich E. Kruse3, Shigeru Kinoshita2, Noriko Koizumi1.
1
Biomedical Engineering, Doshisha University, Kyotanabe, Japan;
2
Ophthalmology, Kyoto Prefectural University of Medicine,
Kyoto, Japan; 3Ophthalmology, University of Erlangen, ErlangenNuremberg, Germany.
Purpose: A tissue engineering technique for treating corneal
endothelial dysfunction has been anticipated as an alternative therapy
to conventional corneal transplantation. As cultivation of human
corneal endothelial cells (HCECs) is difficult and cell density is
rapidly decreased after culture passage, an efficient culture protocol is
needed. We previously reported the culture of laminin-511 (LM-511)
enhanced HCECs (ARVO, 2014). The purpose of this study was to
evaluate the effect of LM511-E8 (the minimal functional form of
LM-511) on the culture of HCECs for clinical application.
Methods: Corneal endothelium was isolated from 3 independent
donor corneas, and then seeded on an LM511-E8 (1.0 μg/cm2)-coated
culture dish and an FNC Coating Mix® (Athena Enzyme Systems) (a
widely used culture substrate)-coated culture dish. Cell density was
evaluated by Image J software after reaching confluency at 4 weeks
of cultivation. Next, HCECs were seeded on an LM511-E8-coated
culture dish or on a non-coated culture dish with culture medium
supplemented with LM511-E8 (2.1nM). The numbers of adhered
HCECs were then evaluated by CellTiter-Glo® Luminescent Cell
Viability Assay after 24 hours. The effect of functional blocking of
integrin α3β1 and α6β1 by neutralizing antibody were then evaluated
by Cell Titer-Glo® and western blotting.
Results: The average cell density of the HCEC culture on LM511-E8
was 2397.1±264.8 cells/mm2, while that on FNC Coating Mix®
was 1203.3±97.9 cells/mm2 (p<0.01). The cell adhesion of HCECs
cultured on the LM511-E8-coated culture dish was significantly
enhanced by 1.3 fold compared with that the non-coating dish
(p<0.01). In addition, cell adhesion was also enhanced by 1.3 fold
when HCECs were seeded with the culture medium supplemented
with LM511-E8 (p<0.01). However, the neutralizing antibody of
integrin α3β1 and α6β1 suppressed the cell adhesion enhanced by
LM511-E8. Phosphorylation of focal adhesion kinase of HCECs was
promoted by LM511-E8, while it was suppressed by the neutralizing
antibody of integrin α3β1 and α6β1.
Conclusions: Our findings indicate that LM511-E8 modulates the
adhesion of HCECs by activating focal adhesion proteins via binding
to integrin α3β1 and α6β1, and enables efficient in vitro expansion of
HCECs with maintaining high cell density for regenerative medicine
for corneal endothelial dysfunction.
Commercial Relationships: Kazuya Kakutani, None; Naoki
Okumura, JCR Pharmaceuticals Co. (P), Senju Pharmaceutical
Co. (P); Ursula Schlotzer-Schrehardt, None; Friedrich E. Kruse,
None; Shigeru Kinoshita, Alcon (R), AMO (R), HOYA (R), Otsuka
Pharmaceutical Co. (C), Santen Pharmaceutical Co. (P), Senju
Pharmaceutical Co. (P); Noriko Koizumi, JCR Pharmaceuticals Co.
(P), Senju Pharmaceutical Co. (P)
Support: Program for the Strategic Research Foundation at Private
Universities from MEXT
Optimization of Human Corneal Endothelial Cell Morphology.
Carlos-Alberto Rodriguez-Barrientos3, Noelia J. Kunzevitzky1, 2,
Alena Bartakova1, Judith Zavala3, Jeffrey L. Goldberg1, Jorge E.
Valdez3. 1Shiley Eye Center, University of California San Diego, La
Jolla, California, San Diego, CA; 2Emmecell, Key Biscayne, Florida,
Miami, FL; 3Instituto Tecnologico de Monterrey, Monterrey, Mexico.
Purpose: Corneal endothelial dysfunction is a leading cause of
blindness and cell therapy approaches are being studied as an
alternative to lamellar and penetrating keratoplasty. Several protocols
for expanding human corneal endothelial cells (HCECs) in vitro
have been developed; however, often HCECs undergo endothelialto-mesenchymal transition and they lose their ability to form a
tight, functional monolayer. Here we describe changes in HCEC
morphology in the presence of various culture media supplements.
Methods: Corneas were dissected, the endothelium was peeled off
and HCECs were isolated and expanded in vitro for at least 2 weeks
in culture medium containing fetal bovine serum (FBS), nerve growth
factor (NGF), epidermal growth factor (EGF) and bovine pituitary
extract (BPE). After exhibiting morphological changes, fibroblastic
HCECs were switched to basal medium supplemented with FBS and
imaged daily for 7 days. Sister cultures were similarly treated and the
expression of surface markers associated with HCEC morphology
was evaluated by flow cytometry.
Results: HCECs cultured in the presence of FBS and exogenous
growth factors underwent endothelial to mesenchymal transition
during passage 3. After switching them to a basal media
supplemented only with FBS, HCECs retained the fibroblastic
morphology for the first 24 hours but at 72 hours they reverted to
a polygonal (canonical) shape. Increased expression of CD56+, a
surface marker associated with HCEC canonical morphology, was
observed 72 hours after the culture media switch.
Conclusions: These preliminary experiments revealed that when
HCECs undergo endothelial to mesenchymal transformation,
canonical morphology may be restored at least 72 hours later by
switching the culture media to a stabilization media containing
FBS. Future studies are aimed at analyzing the time course of this
transition and may address the molecular mechanisms underlying this
phenomenon
Commercial Relationships: Carlos-Alberto RodriguezBarrientos, None; Noelia J. Kunzevitzky, Emmecell (E); Alena
Bartakova, None; Judith Zavala, None; Jeffrey L. Goldberg,
Emmecell (P), Emmecell (S); Jorge E. Valdez, None
Visual function and corneal endothelium analysis in patients with
well-suppressed HIV infection: a matched case-control study
Nazli Demirkaya1, Ferdinand Wit2, 3, Thomas J. Van Den Berg4,
Peter Reiss2, 3, Frank D. Verbraak1. 1Ophthalmology, Academic Med
Ctr Amsterdam, Amsterdam, Netherlands; 2Infectious Diseases,
Academic Medical Center, Amsterdam, Netherlands; 3Amsterdam
Institute for Global Health and Development, Amsterdam,
Netherlands; 4Netherlands Ophthalmic Research Institute,
Amsterdam, Netherlands.
Purpose: To assess visual function and features of the corneal
endothelium in HIV-infected adults by psychophysical tests and
corneal specular microscopy.
Methods: Fifty-four HIV-infected men and 43 HIV-negative control
subjects were enrolled from the prospective AGEhIV cohort study on
age-associated non-communicable co-morbidity in Amsterdam. All
participants underwent a standard ophthalmic examination. Visual
function was assessed by Pelli Robson and Mars contrast sensitivity
charts, C-Quant straylight meter, Lanthony Desaturated Panel D-15,
Rarebit Fovea Test and Frequency Doubling Perimetry. The Topcon
specular microscope SP-1P was used to assess features of the corneal
endothelium in a subset of the study participants (25 HIV-infected
patients and 12 HIV-negative controls). Only right eyes were
included for analysis by multivariable regression models.
Results: Correcting for age, we did not detect significant differences
in visual function between HIV-infected individuals and controls,
with the only exception of a slightly higher straylight value in the
patient group. Corneal endothelium parameters were also similar
between the two groups.
In the patients, we observed significant associations between a longer
duration of HIV-infection and features of the corneal endothelium
consistent with aging (lower endothelial cell density, higher variation
in cell size and an increased average cell size). Patients with a prior
diagnosis of AIDS had a significant larger average cell size than
patients who never had developed AIDS.
Conclusions: Visual function and corneal endothelium parameters
are not significantly different between patients with well-suppressed
HIV-infection and healthy controls, although a longer duration of
HIV is associated with changes in the corneal endothelium consistent
with aging.
Commercial Relationships: Nazli Demirkaya, None; Ferdinand
Wit, None; Thomas J. Van Den Berg, None; Peter Reiss, Bristol
Myers Squibb (F), Merck (F), Institution from Gilead Sciences (F),
Janssen Pharmaceuticals (F), ViiV (F); Frank D. Verbraak, None
Overestimation of Corneal Endothelial Cell Density by Selecting
Smaller Areas to Count the Cells in Images of In Vivo Confocal
Microscopy
Ahmad Kheirkhah, Ujwala Saboo, Reza Dana. Ophthalmology,
Massachusetts Eye and Ear Infirmary, Boston, MA.
Purpose: Laser scanning in vivo confocal microscopy (LS-IVCM) is
commonly used to measure corneal endothelial cell density (CECD).
However, due to corneal applanation during imaging, it is common
to see dark non-focused areas in LS-IVCM images which make it
impossible to count all corneal endothelial cells in the image. It is
unclear whether counting endothelial cells in smaller areas in LSIVCM images will affect the measured CECD.
Methods: 32 endothelial images by LS-IVCM (Heidelberg Retina
Tomograph 3 with Rostock Corneal Module; Heidelberg, Germany)
with different endothelial cell densities were analyzed. In each image
(160 000 mm2), endothelial cells were counted within the following
areas: 80 000, 40 000, 20 000, 10 000, 5 000, and 2 500 mm2. The cell
numbers counted in these areas were then expressed as cell/mm2, and
the CECD were compared.
Results: The mean measured CECD was 2007 ± 750 cells/mm2,
2109 ± 764 cells/mm2, 2149 ± 770 cells/mm2, 2290 ± 750 cells/
mm2, 2495 ± 758 cells/mm2, and 2760 ± 854 cells/mm2 for areas of
80 000 mm2, 40 000 mm2, 20 000 mm2, 10 000 mm2, 5 000 mm2, and 2
500 mm2, respectively; the differences between all these groups were
statistically significant (all P<0.05). Compared to the CECD in 80
000-mm2 area, the mean percentage of overestimation was 6.2 ± 6.7%
for the 40 000-mm2 area, 8.9 ± 11.4% for the 20 000-mm2 area, 18.7
± 21.1% for the 10 000-mm2 area, 27.9 ± 23.6% for the 5 000-mm2
area, and 45.6 ± 34.9% for the 2 500-mm2 area. There were significant
negative correlations between the measured CECD for the 80 000mm2 area and the percentage of overestimation in the smaller areas
(R= -0.44 to -0.71, all P<0.05).
Conclusions: Counting endothelial cells in smaller areas of LSIVCM images results in a significant overestimation of CECD.
Commercial Relationships: Ahmad Kheirkhah, None; Ujwala
Saboo, None; Reza Dana, None
Correlation of retroillumination photography analysis and
clinical severity grading method in Fuchs Corneal Dystrophy
Brian Garrett1, Aisha Mumtaz2, Elyse J. McGlumphy4, Benjamin W.
Iliff4, Armand Edalati3, Simon Ghofrani4, Mahsa Rezaei4, Allen O.
Eghrari4, John D. Gottsch4. 1Dalhousie Medical School, Halifax,
NS, Canada; 2George Washington University School of Medicine,
Washington, DC; 3University of Missouri Kansas City, Kansas City,
MO; 4Johns Hopkins University School of Medicine, Baltimore, MD.
Purpose: Fuchs Corneal Dystrophy (FCD) is a progressive,
hereditary corneal disorder marked by central corneal edema and
the presence of guttae, excrescences of Descemet membrane.
Traditional grading of severity is based on slit-lamp biomicroscopy
and subjective clinical assessment of the distribution of guttae and
presence of corneal edema; however, we have previously shown
that retroillumination photography analysis (RPA) can objectively
and reliably assess progression of disease and distribution of guttae,
which accelerate in their development over time. Here, we explore
the correlation between two methods and implications for high levels
of severity.
Methods: Retroillumination photography was conducted in 54
individuals affected with FCD; all subjects provided written informed
consent. The number of guttae were summated manually. Exclusion
criteria included history of intraocular surgery or inflammation.
Clinical grading using the Krachmer scale measuring 1 to 5 was
documented for each eye at the time of examination. Regression
analyses were performed to identify the best-fit model between the
two methods. Ranges of guttae were assessed at each stage of clinical
grading.
Results: A total of 96 retroillumination photographs passed exclusion
criteria and were analyzed. Krachmer score ranged in severity from 1
to 5, with mean score of 2.625. The mean number of guttae in corneas
at each clinical level of severity (Krachmer grading in parentheses)
were 289 (1+), 999 (2+), 2669 (3+), 5474 (4+), and 7133 (5+).
Higher levels of clinical grading were associated with larger ranges
of guttae (p<0.02), with corneas identified as 5+ demonstrating 7227
guttae between the maximum and minimum affected. A power model
resulted in a strong and greater fit between RPA and Krachmer score
(r2=0.78) than a linear model (r2=0.62).
Conclusions: In this largest study of RPA data and comparison with
subjective clinical grading of FCD severity, RPA correlates well
and demonstrates better resolution of severity at advanced stages of
disease. This method provides objective levels of severity and may
benefit clinicians and researchers who seek to track detailed rates of
progression over time.
Commercial Relationships: Brian Garrett, None; Aisha Mumtaz,
None; Elyse J. McGlumphy, None; Benjamin W. Iliff, None;
Armand Edalati, None; Simon Ghofrani, None; Mahsa Rezaei,
None; Allen O. Eghrari, None; John D. Gottsch, None
Designing an innovative bioreactor destined to improve the
endothelial viability of stored corneas
Gilles Thuret2, 3, Aurélien Bernard2, Tanguy Nangoum-Fosso2, Zhiguo
He2, Chantal Perrache2, Simone Piselli2, Sophie Acquart4, Philippe
Gain2, 1. 1Ophthalmology, University Hospital of St-Etienne, SaintEtienne, France; 2Corneal Graft Biology, Engineering and Imaging
Laboratory, EA2521, Federative Institute of Research in Sciences and
Health Engineering, Faculty of Medicine, Jean Monnet University,
Saint-Etienne, France; 3Institut Universitaire de France, Paris, France;
4
Eye Bank, French Blood Centre, Saint-Etienne, France.
Purpose: Corneal physiology is highly dependent on the intraocular
pressure (IOP) and endothelial functions. Loss of IOP after death
and after corneo-scleral procurement triggers a vicious circle with
stromal swelling causing posterior folds in which endothelial
mortality is increased resulting in impaired barrier functions that
accentuate stromal swelling. We hypothesized that restoration of IOP
after procurement will be key in creating a favourable environment
for long-term corneal storage with preserved endothelial functions
necessary for research works as well as for eye banking
Methods: Using machining of PMMA and polycarbonate, 3D
printing and mecatronic technologies, we designed two bioreactors
(BR) (for human and porcine corneas) and their control units.
Contrary to previous perfusion chambers or artificial anterior
chambers destined for physiology or toxicology studies, we designed
a closed-loop system with transparent windows allowing observation
for both the epithelial and endothelial sides. Sterile sampling sites
were added in the circuit for monitoring physiochemical parameters
as well as sterility without risk of contamination
Results: The BR presented as a sterile disposable cassette comprising
a corneal chamber surrounded by the fluidic system comprising the
storage medium tank (sufficient for 5 weeks), tubing, a pressure
sensor, a micro valve, and a waste. After insertion of the corneoscleral rim, the system was closed and the cassette connected
to its control unit, comprising a peristaltic pump driven by a
microcontroller. The first version could drive 3 to 5 BR. The medium
flow could be adjusted (typically 0.16 mL/hour for human corneas)
and the IOP could be individually adjusted between 10 to 40 mmHg
(for research works). If necessary (for instance when using a storage
medium requiring 5% CO2 to ensure pH stability) the BR could be
installed in a classical CO2 incubator. If necessary, the epithelial
chamber could be filled with air while keeping the IOP
Conclusions: The innovative BR restores IOP while ensuring a
continuous medium renewal in a fully transparent chamber. A closed,
sterile, nearly physiologic environment is actively controlled. The
BR will be available for eye banks, research laboratories, contract
research organizations, and cosmetic industry.
Grants: EFS, ANSM
Commercial Relationships: Gilles Thuret, Quantel Medical (C),
University Jean Monnet (P), University Jean Monnet (P); Aurélien
Bernard, None; Tanguy Nangoum-Fosso, None; Zhiguo He, None;
Chantal Perrache, None; Simone Piselli, University Jean Monnet
(P); Sophie Acquart, University Jean Monnet (P); Philippe Gain,
University Jean Monnet (P), University Jean Monnet (P)
Support: EFS and ANSM
A Descemet’s membrane marker stamp to increase safety and
reproducibility of graft preparation for DMEK surgery
Johannes Menzel-Severing, Theofilos Tourtas, Thomas A. Fuchsluger,
Ursula Schlotzer-Schrehardt, Friedrich E. Kruse. Ophthalmology,
Univ of Erlangen-Nuremberg, Erlangen, Germany.
Purpose: Descemet’s membrane endothelial keratoplasty (DMEK)
has improved visual recovery in patients with corneal endothelial
disease. However, the procedure has received criticism for
jeopardizing donor tissue during graft preparation. Standardization
of graft preparation may support corneal surgeons and eye bank
personnel in learning and safely performing the stripping procedure,
thereby minimizing tissue loss. Here, we propose a novel tool
designed specifically for preparation of Descemet’s membrane (DM)
grafts.
Methods: CNC milling was used to create a blunt trephine-like
instrument that can be centered over the donor cornea. The device
was used to deliberately damage endothelial cells in the corneal
periphery by lowering it onto the cornea, thereby briefly touching
the endothelium. A central 9 mm opening ensured that the 8 mm
graft area plus a 0.5 mm safety margin were spared. Trypan blue
0.06% (VisionBlue®) was used to stain the denuded DM, and graft
preparation was continued by peeling a narrow strip of peripheral
DM using a razor blade as previously described (Kruse et al., Cornea
2011). Transmission electron microscopy (TEM) was used to confirm
structural integrity of the denuded DM.
Results: Use of the marker stamp resulted in the delineation of a
peripheral band of denuded DM which readily stained with trypan
blue. This aided in the visualization of DM during subsequent peeling
steps. The central margin could be easily identified and the risk of
the “rhexis” escaping centrally was reduced. Also, radial tears could
be detected as they occurred and were therefore less likely to remain
unnoticed. TEM confirmed that no structural deficits of DM were
induced by removal of endothelial cells.
Conclusions: Preparation of DM grafts can be further standardized
by providing customized surgical instruments. The device presented
here improves visualization of DM during creation of the peripheral
margin for subsequent lifting of the margin and stripping of the
graft. It thereby offers increased safety and reproducibility and may
also shorten preparation times and learning periods. Damage to
the membrane itself was not observed, suggesting that the ability
to withstand grasping and pulling during DM stripping is not
compromised.
Commercial Relationships: Johannes Menzel-Severing, None;
Theofilos Tourtas, None; Thomas A. Fuchsluger, None; Ursula
Schlotzer-Schrehardt, None; Friedrich E. Kruse, None
A Technique for Quantification of Endothelial Cell Loss in Eye
Bank Prepared DMEK Grafts Intended for Clinical Use
Jeffrey D. Holiman, Philip Dye, Chris Stoeger. Lions VisionGift,
Portland, OR.
Purpose: Descemet membrane endothelial keratoplasty (DMEK) is
a surgical technique to replace dysfunctional corneal endothelium.
Damage to endothelial cells in the graft area can easily occur
during the separation of Descemet membrane (DM) from stroma
while preparing the graft. This damage may affect suitability for
transplant by reducing the endothelial cell density (ECD). Trypan
blue stain 0.06% (DORC, The Netherlands) is currently used in
some preparation protocols to visualize the free edge of DM. This
stain can be used to visualize cells with ruptured cell membranes and
areas of denuded DM. The objective of this study is to determine if
pan-endothelial cell loss (ECL) of the graft area can be determined
conveniently and without compromising graft viability by using the
trypan blue stain. Additionally, a more accurate measurement of ECD
may be determined by combining percent ECL with known ECD as
determined by specular microscopy.
Methods: Six human donor corneas unsuitable for transplant were
prepared for DMEK in an eye bank setting under an operating
microscope, age range 52-73 yrs, median age 54 yrs, with regular
endothelium and avg ECD of 2457 cells/mm2 with range 20123040. Trypan blue staining for 90 seconds was used to visualize
endothelium that lost viability using a comparison of pre-preparation
image to post-preparation image. The central 8mm was digitally
cropped and ECL was calculated using Image J with trainable
segmentation plug-in to obtain a histogram with pixel counts of
area of damage indicated by stain uptake divided by total area of
endothelium in the central 8mm. Final ECD was calculated by
subtracting the % ECL from the ECD.
Results: All six DMEK grafts were able to be analyzed. The average
total cell loss of graft area is 3.88% (range 2.0-5.9%) with the
average net cell loss at 3.2% (range 1.6-5.0%). Final ECD average
was 2365 (range 1906-2920).
Conclusions: In this small series the combined average ECL in 6
corneas prepared for DMEK in an eye bank setting was 3.2%. We
have demonstrated that a more accurate final ECD may be determined
than by specular microscopy alone which is the standard protocol in
US eye banks. This measurement was obtained in a manner that does
not compromise tissue quality.
Commercial Relationships: Jeffrey D. Holiman, None; Philip Dye,
None; Chris Stoeger, None
Scheimpflug Imaging Characteristics of Fuchs Endothelial
Corneal Dystrophy
Katrin Wacker, Jay W. McLaren, Katrina M. Kane, Keith H. Baratz,
Sanjay V. Patel. Department of Ophthalmology, Mayo Clinic,
Rochester, MN.
Purpose: Anterior corneal haze (backscatter), measured by confocal
microscopy, increases in Fuchs endothelial corneal dystrophy
(FECD). In clinical practice, non-contact Scheimpflug cameras
are faster and simpler to use than confocal microscopy. This study
assessed Scheimpflug image characteristics across a range of severity
of FECD.
Methods: Eighty-three eyes of 46 participants (median age, 67
years; range, 44–89 years) were examined by using slit-lamp
biomicroscopy. FECD severity was graded clinically based on the
area and confluence of guttae, and the presence of edema (modified
Krachmer scale). Eyes were grouped as controls (grade 0, n = 16), or
mild (grade 1–2, n = 25), moderate (grade 3–4, n = 24), or advanced
(grade 5–6, n = 18) FECD. Central corneal haze in the anterior 120
μm, mid-stroma, and posterior 60 μm of the cornea was measured
from Scheimpflug images (Pentacam) standardized to a fixed scatter
source and expressed as “Scatter Units” (SU), the concentration of
Amco Clear (GFS Chemicals) that gave the same image brightness
as the corneal image. Effective endothelial cell density (ECDe), an
objective measure of disease severity, was determined from the
area of guttae and local cell density in confocal microscopy images
(ConfoScan 4) of the same corneas. Central corneal thickness (CCT)
was measured by ultrasonic pachymetry. Variables were compared
between groups by using generalized estimating equation (GEE)
models to account for correlation between fellow eyes. Correlations
were assessed by using Pearson coefficients with significances
determined by using GEE models.
Results: Central corneal haze in mild, moderate, and advanced FECD
was higher than normal in all three depths of the cornea (Table).
ECDe was correlated with anterior (r = -0.55, p < 0.001), mid-stromal
(r = -0.43, p = 0.006), and posterior (r = -0.59, p < 0.001) corneal
haze. CCT in mild, moderate, and advanced FECD was higher than
normal (Table). CCT was correlated with anterior (r = 0.41, p <
0.001), mid-stromal (r = 0.31, p < 0.001), and posterior (r = 0.41, p =
0.031) corneal haze.
Conclusions: Scheimpflug imaging detected increased haze at all
depths of the cornea in FECD, and in the earliest stages of FECD.
Haze was associated with increasing disease severity (decreasing
ECDe). Non-contact Scheimpflug photography might provide a
simple method of assessing FECD and its follow-up in clinical
practice.
Purpose: To develop a tissue-engineered human corneal endothelium
by culturing corneal endothelial cells on a human collagen type I
membrane, both isolated from the same donor.
Methods: 1- Donors: Corneal tissue and fresh human cancellous
bone were obtained from the same donor, stored and processed in
Asturias Regional tissue bank according to Spanish laws.
2- Cellular culture: Peripheral endothelium (without trabeculum)
was obtained from corneas during posterior endothelial preparation
(DMEK). Descemet was stripped and trephined, the central part was
used for graft and the peripheral ring was used for tissue engineering.
Peripheral Descemet membrane was cut and the explants were
seeded and cultured on a dish previously treated with fibronectine.
Once confluent, endothelial cells were detached and subcultured on a
collagen type I membrane.
3- Collagen type I isolation: Collagen isolation was performed as
previously described in Sawkins et al. 2013. Briefly, bone granules
were demineralized in HCl 0.5M and lipids were extracted with a
solution of methanol-chloroform. Demineralized bone matrix was
lyophilized and a pepsin digestion and acid solubilization technique
was employed for the isolation of collagen type I. Collagen was
characterized by western blot and quantified using a hydroxyproline
assay.
4- Collagen membranes: 3 mg/cm2 of isolated collagen type I was
cast into a silicone mold and air-dried at room temperature. Collagen
membranes were crosslinked using 254nm UV irradiation lamp to
improve their resistance.
5- Cellular analysis: Human corneal endothelial cells growing on
the collagen membranes were fixed and analyzed by phase contrast
microscopy, scanning electron microscopy and immunocytochemistry
against Na+/K+ and ZO-1.
Results: Type I collagen was isolated from human bone as proven by
Western blot analysis. Concentration determined by hydroxyproline
ratio was used to cast collagen into a membrane that was, later on,
used as a scaffold. Human endothelial cells were able to attach
and grow onto collagen type I membranes. Cells maintained their
morphology and cellular markers, showing positive Na+/K+ and ZO-1
stain.
Conclusions: We have successfully obtained artificial lamellar
endothelial grafts using cells from Descemet peripheral rings and
collagen membranes from bone of the same donor. This strategy
could supply extra endothelial tissue and compensate the deficit of
cadaveric graft for endothelial transplantation.
Commercial Relationships: Alvaro Meana, None; Natalia
Vázquez, None; Manuel Chacón, None; Miguel Naveiras, None;
Paz Cañal, None; Rosana Martínez, None; Jesus Merayo-Lloves,
None
Support: Mutua Madrileña
Commercial Relationships: Katrin Wacker, None; Jay W.
McLaren, None; Katrina M. Kane, None; Keith H. Baratz, None;
Sanjay V. Patel, None
Support: Research to Prevent Blindness; Mayo Foundation
INNOVATIVE USE OF SPECTRALIS OCT
AUTOFLUORESCENCE ON TRACKING EX-VIVO HUMAN
CORNEAL ENDOTHELIAL CELL COLONIES
Gustavo T. Grottone, Jose A. Gomes, Joyce L. Covre, Renata R.
Loureiro, Nicolas C. Pereira. Ophthalmology, UNIFESP/Santa Casa
de Santos, Santos, Brazil.
Purpose: DEVELOP A NEW METHOD TO TRACK ADHESION
AND MIGRATION PATTERNS OF HUMAN CORNEAL
ENDOTHELIAL CELLS AFTER EX-VIVO IMPLANTATION.
Methods: Human corneal endothelial cells previously cultured
in vitro were divided in two groups: exposed or non-exposed to
magnetic nanoparticles. Those cells were used as an attempt to
regenerate our ex-vivo host corneas. Cell colonies were tracked after
A new tissue engineering approach for endothelial grafts
Alvaro Meana1, 2, Natalia Vázquez1, 3, Manuel Chacón1, 3, Miguel
Naveiras1, Paz Cañal4, Rosana Martínez4, Jesus Merayo-Lloves1, 3.
1
Superficie Ocular III, Fundacion de Investigacion Oftalmologica Instituto Oftalmologico Fernandez-Vega, Oviedo, Spain; 2CIBERER
U714, Asturias, Spain; 3Universidad de Oviedo, Oviedo, Spain;
4
Centro Comunitario de Sangre y Tejidos, Oviedo, Spain.
7 days using an scanning laser ophthalmoscope with a excitation
light of 488 nm. Human corneal endothelial cells previously cultured
in vitro were divided in two groups: exposed or non-exposed to
magnetic nanoparticles. Those cells were used as an attempt to
regenerate our ex-vivo host corneas. Cell colonies were tracked after
7 days using an scanning laser ophthalmoscope with a excitation light
of 488 nm.
Results: Human corneal endothelial cells showed no toxicity when
exposed to the studied nanoparticle(p=0.507). Both, non-magnetic
and magnetic cells were found attached to posterior surface of
corneas after 7 days of ex-vivo cultures and visualized by C-SLO
Spectralis autofluorescence mode.
Conclusions: Human corneal endothelial cells from magnetic and
non-magnetic groups attached to corneal posterior surface on ex-vivo
hosts.
Commercial Relationships: Gustavo T. Grottone, None; Jose A.
Gomes, None; Joyce L. Covre, None; Renata R. Loureiro, None;
Nicolas C. Pereira, None
Support: FAPESP
Quantification of Corneal Endothelial Cell Death Due to
Compressive Forces
Manuel A. Ramirez Garcia1, Yousuf Khalifa2, Mark R. Buckley1.
1
Biomedical Engineering, University of Rochester, Rochester, NY;
2
Emory University, Atlanta, GA.
Purpose: Corneal endothelial cell (CEC) loss from surgical
trauma is a critical risk factor for corneal graft failure. However,
quantification of the mechanical forces leading to CEC death has
yet to be performed. In this study, we developed a testing platform
to characterize corneal endothelial cell death as a function of
compressive force.
Methods: Intact corneo-scleral rims were dissected from fresh
porcine eyes. Each rim was incubated in a staining solution of
Calcein-AM and BSS to visualize endothelial cell viability. Central
corneal buttons 8 mm in diameter were punched and immediately
placed in a bath of Propidium Iodide (PI) and BSS to asses cell
death from the dissecting and trephination procedures. Buttons were
submerged for the duration of the experiment. Images were taken
before and after loading.
A custom device utilizing a cantilever beam with a 3 mm stainless
steel ball attached was used to indent the endothelium. Experimental
groups (Sham, Minimum, Maximum Force, n=3 each) were subjected
to peak compressive forces of 0.0 mN, 0.005 mN and 10 mN
respectively. Area of cell death was quantified by counting the nuclei
of PI-stained cells and multiplying by average cell area.
Results: No statistical significance was found before and after
loading in the sham control or minimum force experimental
groups. However, the change in area of cell death in the maximum
force experimental group was significant (p = 0.001, Figure 2).
Qualitatively, Calcein-AM and PI stained images demonstrated
minimal cell loss from dissection and trephination (Figure 1).
Conclusions: With our current testing platform we are capable of
applying compressive forces to the endothelium in the micro and
millinewton range in contact areas comparable to surgical tools
currently used. The absence of cell death in the sham and minimum
force groups indicates we can apply small forces that yield little to
no cell death. The presence of significant CEC death in the maximal
force group reveals that a critical threshold force exists within the
force range tested. Identification of this threshold force may be useful
to optimize surgical methods and increase corneal transplant graft
survival rates by minimizing endothelial cell loss through mechanical
trauma.
Representative specimen before and after indentation for the maximal
force (10 mN) group. Calcein-AM (green) stains live cells while PI
(red) stains dead cells.
Error bars represent SEM
Commercial Relationships: Manuel A. Ramirez Garcia, None;
Yousuf Khalifa, None; Mark R. Buckley, None
Endothelial Cell Density in Diabetic and Non-diabetic Corneas: A
Retrospective Eye Bank Study
Yicheng Chen1, Patrick Gore2, Mitchell D. McCartney2, David
C. Gritz1, Prajbot Channa1. 1Ophthalmology, Albert Einstein/
Montefiore, Bronx, NY; 2Lions Eye Institute for Transplant and
Research, Tampa, FL.
Purpose: There is conflicting evidence regarding the effect of
diabetes mellitus on the corneal endothelium. One hypothesis is
that oxidative and osmotic stress from excessive activation of the
polyol pathway and accumulation of sorbitol may contribute to
corneal endothelial damage in patients with diabetes mellitus. In
order to better understand the relationship between diabetes and
corneal endothelial cell changes, we performed a retrospective study
comparing endothelial cell density in diabetic and non-diabetic
corneas from an eye bank database.
Methods: De-identified cornea donor data from July 2013 to June
2014 was supplied by the Lions Eye Institute for Transplant and
Research. Pseudophakic donors were excluded from the study.
Diabetic and non-diabetic donors were distinguished based upon
medical history as well as medication lists. Statistical analysis was
performed using two-tailed Mann-Whitney U tests.
Results: A total of 1486 diabetic corneas and 2488 non-diabetic
corneas were analyzed. The mean age of the diabetic group was 57.27
and the mean age of the non-diabetic group was 53.23. The median
endothelial cell density was 2566 cells/mm2 and 2608 cells/mm2
respectively. The U was 1728931 and the p-value was statistically
significant at 0.0006. When the data was stratified by age into
0-20, 21-30, 31-40, 41-50, 51-60, 61-70, and 71-80 age groups, the
difference between the two populations was no longer statistically
significant with p-values of 0.365, 0.100, 0.283, 0.518, 0.067, 0.576,
and 0.331.
Conclusions: Although initial analysis indicated a statistically
significant difference in endothelial cell density between diabetic
and non-diabetic corneas, age-stratified groups did not reveal
a statistically significant difference. The discrepancy between
the overall results and the age-stratified results may be due to a
different distribution of ages between the diabetic and non-diabetic
groups. The aldose reductase pathway may contribute to corneal
endothelial changes in diabetes, but the results of this study suggest
that endothelial cell density is not significantly affected. However,
other measures of endothelial cell health such as pleomorphism and
polymegathism may differ between diabetics and non-diabetics.
Table 1. Age-stratified groups did not show a statistically significant
difference in endothelial cell density between the two populations.
Commercial Relationships: Yicheng Chen, None; Patrick Gore,
None; Mitchell D. McCartney, None; David C. Gritz, None;
Prajbot Channa, None
Corneal endothelial parameters as biomarkers for diabetes in
children
Allison Hinko, David Rogers, Nick F. Fogt. Ohio State University,
Columbus, OH.
Purpose: To assess endothelial cell morphology in type I diabetics
using a non-contact specular microscope and to compare the
endothelial cell morphology to age-matched controls. To compare
changes in endothelial cell morphology in type I diabetics to
traditional measures of diabetic control.
Methods: A total of 25 diabetic children were recruited from the eye
clinics at Nationwide Children’s Hospital (NCH) from March 2012 to
April 2014. Photographs of the corneal endothelium of each eye were
obtained via the Konan SP-9000 non-contact specular microscope.
The Konan KSS-300 software was then used to calculate endothelial
parameters including the coefficient of variation (COV), percentage
of hexagonal cells (%Hex) and the endothelial cell density (ECD).
Comparisons of the mean values of endothelial parameters were
made between diabetic and control groups. Correlations were made
for the diabetic subjects between the COV and the duration since
diagnosis, the %Hex versus the duration since diagnosis, the COV
and the HbA1C, and the %Hex versus HbA1C.
Results: There was no significant difference in the mean age of type
I diabetic subjects (12.60 ± 3.44) versus that of the control group
(11.74 ± 2.85). In the diabetic group, the mean corneal thickness
(585.3 ± 27.7) and endothelial cell density (2945 ± 350) were
not significantly different from those values of the control group
(578 ± 43.1 and 3018 ± 322, respectively). There was, however, a
significant difference between the 2 groups in regards to the COV
and the %Hex. The COV in the diabetic group was 30.75 ± 4.77,
which was significantly higher than that of the controls (25.05 ±
3.42). The %Hex in the diabetic group was (66.68 ± 8.14), which was
significantly lower than that of the controls (72.24 ± 7.30). Within
the diabetic group, there was no linear relationship between the
COV or the %Hex and the HbA1C. There was, however, a moderate
relationship between the COV and duration of diabetes as well as the
%Hex and the duration of diabetes.
Conclusions: The results of this study suggest that in diabetic
children the %Hex decreases. The COV was significantly higher in
the diabetic group. The rest of the parameters were not significantly
different. These data suggest that the corneal endothelium of diabetic
children is morphologically different from that of non-diabetic
children. The results demonstrated that the longer the duration of
diabetes, the lower the %Hex and the higher the COV.
Commercial Relationships: Allison Hinko, None; David Rogers,
None; Nick F. Fogt, None
Comparison of corneal specular microscopy features in pediatric
glaucomatous eyes versus non-glaucomatous eyes
Sahar Bedrood, Dilshad Contractor, Xuejuan Jiang, Bibiana J.
Reiser. Ophthalmology, USC Eye Institute, Los Angeles, CA.
Purpose: In order to understand the pathophysiology of glaucoma
in pediatric patients, features of the ocular anatomy and the cornea
need to be better understood. Specular microscopy is a non-invasive
technique to assess the structure and function of the corneal
endothelium. The aims of this study were to study the specular
microscopy features of pediatric eyes with glaucoma and compare
them to normal pediatric eyes.
Methods: We performed a cross-sectional study of 47 patients (76
eyes) between the ages of 0-18 years with a diagnosis of glaucoma
in at least one eye. The exclusion criteria was corneal pathology and
narrow angle glaucoma. Specular microscopy was performed either
in clinic or under EUA as apropriate. The endothelial cell density,
hexagonality of the cells, corneal thickness and variability of the
cell density were compared. The endothelial cell density analysis
was performed manually using the center to center technique with
the Konan Specular Microscope (Konan Medical USA., Torrance,
CA) along with the KSS-300 analysis software. The comparisons
between glaucomatous and normal eyes were tested using generalized
estimating equations adjusting for participant’s age and between-eye
correlation.
Results: The average subject age was 9 years and 56% were male. 51
eyes had glaucoma and 26 eyes were non-glaucomatous. Endothelial
cell density was significantly lower in glaucomatous eyes (2691
cells/mm2) than normal eyes (3188 cells/mm2) (P=0.005). The
percentage of hexagonal cells was marginally significantly lower in
glaucomatous eyes (67.2%) than normal eyes (71.8%) (P=0.080). The
corneal thickness shows no significant difference between normal
eyes (577 +/- 10) and glaucomatous eyes (570+/-11). However, the
coefficient of variation of the cells was found to decrease with age
(p=0.046) and the total number cells increased with age (p=0.024).
Conclusions: The corneal endothelial cell density of pediatric
patients with glaucoma is lower than in patients with nonglaucomatous eyes. However, the hexagonality of the cells and the
corneal thickness do not differ significantly between the two groups.
While causality can not be implied from this study, these results
indicate an anatomical difference between the corneas of the two
groups and provide information regarding the anatomical differences
of the cornea in pediatric patients with glaucoma.
Commercial Relationships: Sahar Bedrood, None; Dilshad
Contractor, Allergan (F); Xuejuan Jiang, None; Bibiana J. Reiser,
Allergan (F)
Support: Allergan Sales, Inc
Characterization of cryopreserved primary human corneal
endothelial cells cultured in human serum supplemented media
Lucas Vianna1, 2, Hao D. Li1, Jeffrey D. Holiman3, Chris Stoeger3,
Rubens Belfort4, Albert S. Jun1. 1Ophthalmology, Johns Hopkins
University, Baltimore, MD; 2Ophthalmology, Federal University of
São Paulo and State University of Rio de Janeiro, São Paulo, Brazil;
3
Lions VisionGift, Portland, OR; 4Ophthalmology, Federal University
of São Paulo, São Paulo, Brazil.
Purpose: The use of human serum supplemented media (HS-SM)
for human corneal endothelial cell (HCEC) culture has been recently
reported by our group, with potential advantages over fetal bovine
serum supplemented media (FBS-SM) towards making cell therapy
feasible for endothelial diseases in clinical practice. The maintenance
of endothelial cell characteristics after cryopreservation would be
another important consideration regarding this approach. This study
compares cryopreserved HCECs grown in HS-SM to cryopreserved
HCECs grown in FBS-SM.
Methods: Three pairs of human corneas from donors aged 8, 28,
and 31 years old were obtained from Lions VisionGift (Portland,
OR). From each pair, one cornea was used to start a HCEC culture
with HS-SM and the other cornea with FBS-SM. Upon reaching
confluence (P0), the 6 cell populations were frozen using 10%
dimethyl sulfoxide containing medium. Thawed cells grown in
HS-SM were compared to thawed cells grown in FBS-SM by
morphology, growth curve, and protein (immunohistochemistry)
and gene expression (real time-reverse transcriptase polymerase
chain reaction - RT-PCR) for zonula ocludens-1 (ZO-1, gene TJP1),
sodium/potassium ATPase (Na+/K+-ATPase, gene ATP1A1) and
glypican 4 (GPC4, gene GPC4).
Results: No difference in morphology could be seen in P0, P1, P5 or
in any other passages for cells grown in the two media before or after
cryopreservation. By growth curves, cell counts after thawing were
similar in FBS-SM and HS-SM from days 1 to 10, with a slight trend
toward higher cell counts in FBS-SM. Cells grown in both FBS-SM
and HS-SM media showed similar expression of endothelial cell
markers when assessed by immunohistochemistry, although the gene
expression of HCEC markers was higher in HS-SM when assessed by
RT-PCR: TJP1 (1,3-fold, p=0,083), ATP1A1 (2-fold, p= 0,031) and
GPC4 (3-fold, p=0,064).
Conclusions: After cryopreservation, HS-SM was similar to FBS-SM
for HCEC culture when assessed by cell morphology, proliferation,
and protein expression. Marker gene expression by RT-PCR was
higher in HS-SM compared to cells grown in FBS-SM.
Commercial Relationships: Lucas Vianna, None; Hao D. Li,
None; Jeffrey D. Holiman, None; Chris Stoeger, None; Rubens
Belfort, None; Albert S. Jun, None
Wound Healing of Corneal Endothelial Cells from Periphery
After Chemical Injury
Young Joo Shin1, Jin Joo Kim3, Joon-Young Hyon2, Won Ryang Wee2.
1
Ophthalmology, Hallym University College of Medicine, Seoul;
2
Seoul National University College of Medicine, Seoul, Korea
(the Republic of); 3Seoul National University Bundang Hospital,
Seongnam, Korea (the Republic of).
Purpose: To investigate the wound healing process of corneal
endothelial cells (CECs) from chemically induced corneal endothelial
injury.
Methods: The anterior chambers of eyes from New Zealand white
rabbits were injected with 0.1 mL of 0.05 N NaOH for 10 min
(NaOH group). Corneal edema was evaluated at day 1, 7, and 14 after
endothelial injury. Vital staining was performed using alizarin red and
trypan blue.
Results: In the animal study, corneal edema was greater in the NaOH
group compared to that in the control group at both day 1 and day
7. At day 1, no CECs were detected in the center and the periphery
of the corneas in the NaOH group. One week after injury, large and
non-hexagonal CECs were detected in peripheral corneas, but no
cells were detected in central corneas. Two weeks after injury, small,
hexagonal CECs were detected in the peripheral cornea, while in the
mid-periphery, CECs were large and non-hexagonal.
Conclusions: CECs migrated from the periphery to the center of the
cornea after endothelial injury. The peripheral corneal endothelium
may act as a cell reservoir for the recovery of corneal endothelium.
Commercial Relationships: Young Joo Shin, None; Jin Joo Kim,
None; Joon-Young Hyon, None; Won Ryang Wee, None
Support: This study was supported by the National Research
Foundation (NRF) grant (2012R1A1A2040118) funded by the Korea
government (MEST).
Interactions of Corneal Endothelial Cells with Stromal Cells
during Corneal Endothelial Injury
Young Bok Lee1, JinJoo Kim3, Joon Young Hyon3, 2, Eui-Sang Chung4,
Tae-Young Chung4, Won Ryang Wee2, Young Joo Shin1. 1Department
of Ophthalmology, Hallym University College of Medicine,
Seoul, Korea (the Republic of); 2Department of Ophthalmology,
Seoul National University College of Medicine, Seoul, Korea
(the Republic of); 3Department of Ophthalmology, Seoul National
University Bundang Hospital, Seongnam, Korea (the Republic of);
4
Sungkyunkwan University School of Medicine, Samsung Medical
Center, Seoul, Korea (the Republic of).
Purpose: To investigate the interaction of corneal endothelial
cells (CECs) with corneal stromal cells (CSCs) on the healing of
chemically induced corneal endothelial injury.
Methods: New Zealand white rabbits were injected with 0.1 mL of
0.05N NaOH into the anterior chamber for 10 min. Corneal edema
and thickness were evaluated at day 1, 7, 14 and 28 after endothelial
injury. Vital staining with alizarin red and trypan blue was performed.
Immunofluorescein staining of collagen VIII alpha2 (COL8A2),
glial fibrillary acidic protein (GFAP), and alpha-smooth muscle
actin (α-SMA) and deoxynucleotidyltransferase (TdT)-mediated
dUTP nick end labeling were performed. Human corneal endothelial
cells (HCECs) and CSCs were co-cultured in a Transwell system.
Concentration of transforming growth factor-beta1 (TGF-β1)and
interleukin-6 (IL-6)in the media was measured by an enzyme-linked
immunosorbent assay. Intracellular expression of COL8A2 were
evaluated by western blotting.
Results: Corneal edema and corneal opacity scores were higher in
all rabbits exposed to NaOH than in controls. Corneal endothelial
apoptosis with minimal damage to CSCs was observed in rabbits
exposed to NaOH at day 1, and CECs migrated from the periphery.
Two weeks after injury, CSCs were aligned in parallel to the
Descemet’s membrane, which exhibited the presence of CECs.
TGF-β1and IL-6 secretion was lower in the co-culture system than
inindividual cultures of HCECs or CSCs. COL8A2 expression was
higher in HCECs co-cultured with CSCs compared to the HCECs
only without CSCs.
Conclusions: Intracameral NaOH injection is a model of
corneal endothelial injury with minimal impact on the stroma.
Communication between CECs and CSCs during wound healing may
lead to the modulation of cytokine production, which is necessary for
an enhanced healing process of corneal endothelial injury.
Commercial Relationships: Young Bok Lee, None; JinJoo Kim,
None; Joon Young Hyon, None; Eui-Sang Chung, None; TaeYoung Chung, None; Won Ryang Wee, None; Young Joo Shin,
None
Predicting endothelial cell loss during organ culture of the human
cornea using morphological parameters
Martin Hermel, Matthias Fuest, Sabine Salla, Peter Walter.
Ophthalmology, RWTH Aachen University, Aachen, Germany.
Purpose: Endothelial assessment is a critical step in the release
of corneas for grafting. In addition to endothelial cell density
(ECD), further criteria are applied to assess the morphology of
the endothelium. We performed a retrospective study to determine
the role of endothelial morphological parameters in predicting
endothelial cell loss during organ culture.
Methods: Human donor corneas were harvested and placed in
minimal essential medium with 2% fetal calf serum, penicillin,
streptomycin and amphotericin B. At the beginning of culture,
endothelial morphology was assessed microscopically using the
parameters polymegatism (POL), pleomorphism (PLE), granulation
(GRA), vacuolization (VAC), segmentation of cell membranes
(SEG), Descemet’s folds (DF), trypane blue positive cells (TBPC)
and endothelial cell-free areas (ECFA), using standardized scores
from 0 to 3. Some corneas were primarily rejected based on
endothelial assessment. ECD was assessed at the beginning (I-ECD)
and end of culture. Corneas were then placed in dehydration medium
(as above + 5% dextran 500). In a subgroup, ECD was reassessed
after dehydration. Endothelial cell loss during culture (ECL-C)
and culture + dehydration (ECL-D) were calculated. Statistical
analyses were performed using multiple linear regression and logistic
regression, as appropriate (SAS/JMP11). Odds ratios (OR) were
expressed per parameter level.
Results: At initial assessment ECD was 2811+361/mm2 (n=2362).
The decision to reject a cornea due to endothelial assessment was
associated (r2 = 0.1760, p< 0.0001) with ECFA (p< 0.0001, OR
= 2.7), SEG (p< 0.045, OR =1.3), I-EDC (p<0.0001, OR 0.997)
and donor age (p<0.0001, OR 1.024). ECL-C was 153+200/mm2
(n=1277), ECL-D was 169+183/mm2 (n=918) with a culture duration
of 19.71+6.5 days. ECL-C was associated (r2 = 0.081, p<0.0001)
with DF (p<0.0001), TPBC (p=0.0011), GRA (p=0.0022), PLE
(p=0.03), I-EDC (p<0.0001) and donor age (p<0.0053). ECL-D was
associated (r2 = 0.228, p< 0.0001) with TPBC (p=0.0019), VAC
(p=0.0003), PLE (p=0.0015), POL (p=0.0064), I-EDC (p<0.0001),
donor age (p<0.0079) and gender (p=0.022).
Conclusions: Several morphological parameters displayed significant
predictive value for a primary exclusion of the corneas from culture
and a significant correlation with endothelial cell loss. The selection
of appropriate parameters may help to improve graft quality.
Commercial Relationships: Martin Hermel, None; Matthias
Fuest, None; Sabine Salla, None; Peter Walter, None
Investigation of the donor tissue information on the phenotypes
of cultivated human corneal endothelium
Hiroshi Tanaka1, Morio Ueno1, Munetoyo Toda1, Junji Hamuro2,
Kengo Yoshii2, Noriko Koizumi3, Naoki Okumura3, Shigeru
Kinoshita1, Monty Montoya4, Bernie Iliakis4. 1Ophthalmology, Kyoto
Prefectural University of Medicine, Kyoto, Japan; 2Medical Statics,
Kyoto Prefectural University of Medicine, Kyoto, Japan; 3Biomedical
Engineering, Doshisha University, Kyotanabe, Japan; 4Sight Life,
Seattle, WA.
Purpose: We are developing a novel technology for corneal
regenerative medicine for bullous keratopathy (BK) that involves the
injection of cultivated human corneal endothelial cells (cHCECs) into
the anterior chamber. One key for a successful corneal transplantation
is the preparation of fully differentiated cHCECs with a cobblestonelike shape. However, mature cHCECs with a fully differentiated
phenotype are damped under in vitro culture conditions by
phenotypic transition; i.e., epithelial-mesenchymal transition (EMT),
cell senescence, and fibrosis. The purpose of this present study was
to clarify the relationship between this phenotypic transition and the
characteristics of donor human corneal tissues.
Methods: Donor human corneal tissues were obtained from an eye
bank (SightLife, Seattle, WA) and HCECs obtained from individual
donor corneas were re-suspended. Basal growth medium containing
OptiMEM®-I Reduced Serum Medium (Life Technologies), fetal
bovine serum, epidermal growth factor, ascorbic acid, calcium
chloride, chondroitin sulfate, and gentamicin was conditioned by
cultivation of human bone marrow-derived mesenchymal stem cells
(MSCs), and then recovered as culture medium for HCECs. All cells
were cultured using the MSC-conditioned basal growth medium
supplemented with Rho kinase inhibitor and TGF-β inhibitor.
Proportions of fully differentiated cHCECs after primary culture or 2
serial passage culture were detected using flow cytometry according
to a combination of several cell surface markers. We examined the
relationship between the percentages of fully differentiated cHCECs
and donor information including gender, age, race, cause of death,
and CEC density.
Results: This study included 39 donors (27 males, 12 females;
mean age: 42.2±22.7 years). Mean CEC density was 3105.5±315.3/
mm2. Races were grouped into Caucasian or other. Cause of death
was classified into acute disease, chronic disease, or other. The
percentages of differentiated cHCECs had relation to donor age
(R=-0.60, p=0.0002) and CEC density (R=0.42, p=0.009). CEC
density was also related to age (R=-0.44, p=0.007). The percentages
of differentiated cHCECs had no relation to gender (p=0.81), race
(p=0.76), and cause of death (p=0.16).
Conclusions: The percentage of mature cHCECs with fully
differentiated phenotype is related to donor age and CEC density.
Commercial Relationships: Hiroshi Tanaka, None; Morio Ueno,
Santen Pharmaceutical Co (P), Senju Pharmaceutical Co (P);
Munetoyo Toda, None; Junji Hamuro, None; Kengo Yoshii, None;
Noriko Koizumi, Doshisha University (P), JCR Pharmaceuticals
Co (P), Senju Pharmaceutical Co (P); Naoki Okumura, Doshisha
University (P), JCR Pharmaceuticals Co (P), Senju Pharmaceutical
Co (P); Shigeru Kinoshita, Otsuka Pharmaceutical Co (P), Santen
Pharmaceutical Co (P), Senju Pharmaceutical Co (P); Monty
Montoya, Sight Life (S); Bernie Iliakis, Sight Life (S)
Culture, functional characterization and stability of human
corneal endothelial cells
Noelia J. Kunzevitzky2, 1, Alena Bartakova2, Katarzyna Wilczek3,
Jonathan Sheu3, Jonathan Van Dyke4, Gerhard Bauer3, Jeffrey L.
Goldberg2. 1Emmecell, Key Biscayne, FL; 2Shiley Eye Center,
University of California San Diego, La Jolla, CA; 3Stem Cell
Program, University of California Davis, Sacramento, CA; 4Flow
Cytometry Core, University of California Davis, Sacramento, CA.
Purpose: Corneal endothelial dysfunction is a leading cause of
blindness. Surgical treatment is limited by scarcity of donor tissue
and cost. Cell therapy could overcome these limitations. Several
methods have been developed for human corneal endotheial cell
(HCEC) culture; however, with increased number of passages,
HCECs undergo endothelial-to-mesenchymal transition (EnMT)
and lose function. Here we present a method that stabilizes HCEC
morphology and renders functional cells after several passage which
can be shipped for use in cell therapy.
Methods: HCECs isolated from cadaveric donor corneas were
expanded in vitro following the Joyce method (2004). At confluence,
HCEC morphology was assessed by microscopy, identity by flow
cytometry and RT-PCR, and function by the trans-epithelial electrical
resistance (TEER) assay. HCECs were next subcultured using either
proliferative or stabilization growth media, or a serial combination of
them. HCEC morphology, identity and function were compared daily
for 7 days. A fraction of the cells were re-suspended in BSS+ and
shipped at 40C. Cell viability, morphology, identity and function were
assessed for 7 days upon arrival.
Results: HCECs underwent EnMT after 3 to 4 passages. Switching
to stabilization medium before each passage helped HCECs maintain
their morphology, identity and function over time.
When HCECs cultured in stabilization medium were exposed to
proliferative medium, HCECs became fibroblastic, and exhibited
a decreased expression of canonical markers and function. Finally,
HCECs transported in 1ml syringes with BSS+ were viable (>70%)
for up to 96 hours when shipped and stored at 4°C, expressed
canonical HCEC markers and maintained their barrier function.
Conclusions: In later passages, HCECs undergo endothelial-tomesenchymal transition. Modifications to the traditional HCEC
culture protocol to include a stabilization step prior to each passage
helps maintain the HCECs’ canonical morphology, identity and
function. Thus, these novel HCEC culture and shipping methods may
be an important milestone towards successful HCEC manufacturing
for clinical use.
Commercial Relationships: Noelia J. Kunzevitzky, Emmecell
(E), Emmecell (P); Alena Bartakova, None; Katarzyna Wilczek,
Emmecell (F); Jonathan Sheu, Emmecell (F); Jonathan Van Dyke,
Emmecell (F); Gerhard Bauer, Emmecell (F); Jeffrey L. Goldberg,
Emmecell (P), Emmecell (S)
Support: NEI P30- EY022589 to UCSD and an unrestricted grant
from Research to Prevent Blindness, Inc.
Optimizing the dual media culture system of propagating human
corneal endothelial cells using GMP-certified reagents
Gary S. Peh1, 2, Benjamin L. George1, Khadijah Adnan1, Xin Yi
Seah1, Heng Pei Ang1, Jodhbir S. Mehta1, 2. 1Singapore Eye Research
Institute, Singapore, Singapore; 2Duke-NUS Graduate Medical
School, Singapore, Singapore.
Purpose: For clinical translation of cell therapy, culture protocol
must be standardized and adhered to strict Good Manufacturing
Practices (GMPs) guidelines. The main aim of this study is to
systematically optimize each GMP-certified component required in
the dual media culture system used for the propagation of human
corneal endothelial cells (CEnCs).
Methods: Pairs of donor corneas deemed unsuitable for
transplantation were procured for this study. For the digestion of
Descemet’s membrane (DM), strips of CEnCs-DM peeled from
donor-matched corneas were incubated in either Collagenase (left)
or Liberase (right). For all other comparative studies, CEnCs were
isolated and expanded using the dual media approach to the second
or third passage to obtain sufficient cell numbers. The experimental
parameters evaluated were: 1) cell attachment; 2) serum used in
culture; 3) single-cell dissociation; and 4) cryopreservation. Results
obtained include assessment of cell morphology, overall cell yield
(manual cell-counts), cell adherence (xCelligence), cell proliferation
(Click-iT EdU) and cell viability (Annexin V / PI) as outlined in
Figure 1.
Results: The assessments of cGMP components substituted into
each of the processes were compared to current reagent used.
Specifically, digestions of DM (n=5) using Liberase was equivalent
to Collagenase. Attachment of CEnCs (n=3) was significantly
improved (p<0.05) when rLaminin 511 was used. Nevertheless, the
use of human collagen coating was comparable to FNC coating mix.
Serum comparison (n=3) revealed that cultures of CEnCs became
inconsistent in the presence of human serum whereas results using
Equalfetal showed comparable cell yield similar to CEnCs grown
in the presence of FBS. Next, cellular dissociation of CEnCs (n=5)
were comparable between TrypLE Express and TrypLE Select.
Finally, for cryo-preservation of CEnCs (n=3), cells cryo-preserved
in CryoScarless were significantly more viable than the three other
freezing medium compared (p<0.05).
Conclusions: The various assessments of cGMP components
substituted into each of the processes as compared to current reagent
used have shown their usability towards clinical translation. Future
in vivo studies are required to substantiate our current findings, an
essential undertaking towards clinical cell therapy.
Figure 1. A schematic of the processes, reagent trialed, and the assays
performed.
Commercial Relationships: Gary S. Peh, None; Benjamin L.
George, None; Khadijah Adnan, None; Xin Yi Seah, None; Heng
Pei Ang, None; Jodhbir S. Mehta, None
Support: This research was supported by the Singapore National
Research Foundation under its Translational and Clinical Research
(TCR) Programme (NMRC/TCR/008-SERI/2013)
Purification of high cell density cultured corneal endothelial cells
by density-gradient centrifugation
Ayaka Kusakabe1, Naoki Okumura1, 2, Hiroatsu Hirano1, Noriko
Koizumi1, Shigeru Kinoshita2. 1Biomedical Engineering, Doshisha
University, Kyotanabe, Japan; 2Department of Ophthalmology, Kyoto
Prefectural University of Medicine, Kyoto, Japan.
Purpose: We have initiated clinical research of a cultured corneal
endothelial cells (CECs) injection therapy for treating corneal
endothelial dysfunction. Cell density (CD) of CECs tends to decrease
during serial passages for expansion culture, though transplantation
of high CD–CECs is thought to be beneficial for the clinical outcome.
The purpose of this study was to investigate the feasibility of densitygradient centrifugation to remove low CD–CECs and to purify high
CD-CECs for clinical application.
Methods: A primary culture of human CECs (HCECs) obtained
from donor corneas was passaged 5–6 times and then used for the
experiments. The cultured HCECs were suspended in Opti-MEM®
Reduced Serum Medium (Life Technologies), and OptiPrep™Density
Gradient Medium was then added. After centrifugation at 800 g for
15 minutes, HCECs were recovered from the pellet and supernatant,
respectively. The diameters of the HCECs were evaluated by flow
cytometry. HCECs recovered from both the pellet and supernatant
were cultured for 4 weeks, and CD and cell area were evaluated by
Image J software. Expression of function-related proteins such as
ZO–1 and Na+/K+–ATPase was examined by immunocytochemistry.
Results: Low CD–CECs were observed as a mosaic pattern in high
CD–CECs after 3 passages. The mean diameter of pellet-HCECs
was significantly smaller than that of supernatant-HCECs (42.9±0.17
μm and 49.5±0.31 μm,) (p<0.01). After 4–weeks culture, the mean
CD of the pellet–HCECs and supernatant–HCECs was 1584.5±69.5
cells/mm2 and 827.8±394.1 cells/mm2 , respectively (p<0.01). The
mean cell area of the pellet–HCECs (interquartile range) was lower
than that of supernatant–HCECs (301.9 (244.6-355.2) μm2 and
635.0 (365.6–1354.6) μm2, respectively). Immunocytochemistry
showed that pellet–HCECs express hexagonal morphology and ZO-1
and Na+/K+-ATPase on their plasma membranes, while hexagonal
morphology and expression of ZO-1 and Na+/K+–ATPase were
partially disrupted in supernatant–HCECs.
Conclusions: Our findings indicate that density–gradient
centrifugation enables the purification of high–CD HCECs by
removing low–CD HCECs. Though a protocol for clinical application
needs to be optimized, density-gradient centrifugation is useful to
purify high–CD HCECs for tissue engineering therapy for corneal
endothelial dysfunction.
Commercial Relationships: Ayaka Kusakabe, None; Naoki
Okumura, Doshisha University (P), JCR Pharmaceuticals Co (P),
Senju Pharmaceutical Co (P); Hiroatsu Hirano, None; Noriko
Koizumi, Doshisha University (P), JCR Pharmaceuticals Co. (P),
Senju Pharmaceutical Co. (P); Shigeru Kinoshita, Alcon (R),
AMO (R), HOYA (R), Otsuka Pharmaceutical Co (C), Santen
Pharmaceutical Co (P), Senju Pharmaceutical Co (P)
Matrix metalloproteinase inhibitor reverses endothelialmesenchymal transformation
Wei-Ting Ho1, 2, I-Jong Wang3, 4. 1Ophthalmology, Far Eastern
Memorial Hospital, New Taipei City, Taiwan; 2Gradualte institute
of clinical medicine, National Taiwan University, Taipei, Taiwan;
3
Ophthalmology, National Taiwan University Hospital, Taipei,
Taiwan; 4College of Medicine, National Taiwan University, Taipei,
Taiwan.
Purpose: Ex vivo culture of human corneal endothelial cells is
often subjected to gradual mesenchymal transformation and loss
of function. The present study was conducted to demonstrated the
effect of matrix metalloproteinase inhibitor (MMPI) in reversing
endothelial-mesenchymal transformation (EnMT) during ex vivo
culture and in vivo.
Methods: Bovine corneal endothelial cells (BCECs) were cultured in
medium with or without MMPI. Cell shape and protein localization
were shown by immunofluorescence. MMP activity was monitored
by fluorogenic substrate cleavage assay. Real-time PCR was used
to determine RNA expression, and protein level was determined by
Western blot. To determine the EnMT-reversing effect of MMPI
in vivo, rat corneal endothelium cryo-injury model was used.
Subsequent ultrasound biomicroscopy and histological examination
were performed to evaluate corneal thickness.
Results: During ex vivo culture, BCECs underwent EnMT and
had upregulated MMP expression. Addition of MMPI to cultured
BCECs decreased the level of EMT regulators, snail and slug. The
phosphorylation and degradation of active beta-catenin was also
accelerated after MMPI, which may result from decreased N-cadherin
shedding and increased N-cadherin level on the cell membrane. In
rat corneal endothelium cryo-injury model, intracameral injection of
MMPI reversed EnMT and resulted in less corneal edema.
Conclusions: MMPI can reverse EnMT and preserve the function of
corneal endothelial cell both during ex vivo expansion and in vivo.
This may offer a therapeutic target in regenerative medicine for the
treatment of corneal endothelial dysfunctions.
Commercial Relationships: Wei-Ting Ho, None; I-Jong Wang,
None
Engineered FGF-1 derivatives stimulate proliferation of corneal
endothelial cells
David Eveleth1, Xue Xia2, Ken Thomas1, Michael Blaber2. 1Trefoil
Therapeutics LLC, San Diego, CA; 2Dept of Biomedical Sciences,
College of Medicine, Florida State University, Tallahassee, FL.
Purpose: Fibroblast growth factors (FGFs) are potent stimulators
of corneal endothelial cell growth in vitro and in vivo, but their
therapeutic value is compromised due to poor pharmaceutical
properties. We sought to improve the stability and potency of FGF-1
via site-directed mutagenesis and evaluate the potency and suitability
of these modified compounds for stimulation of corneal endothelial
cell growth.
Methods: Site-directed mutagenesis was used to generate
engineered FGF-1s (eFGF-1s) with enhanced stability and improved
pharmaceutical properties. Biophysical and structural properties
were measured using isothermal equilibrium denaturation and
X-ray crystallography. Pharmacokinetics were evaluated in rabbits
using ELISA-based quantification of FGFs. Mitogenic activity
was evaluated using primary cultures of rabbit and human corneal
endothelial cells.
Results: Substitutions at positions 12, 117 and 134 resulted in
an increase in melting temperature of 19K and a 33-fold increase
in activity in the absence of added heparin. Substitutions of the
cysteines at positions 83 and 117 are thermodynamically destabilizing
but can be compensated for by stabilizing mutations at positions 44
and 132. eFGF-1s exhibit longer half-lives in rabbits and stimulate
mitogenesis of primary rabbit and human corneal endothelial
cells at EC50 values substantially lower than wtFGF-1, even in the
absence of heparin. Cultures maintained a morphology consistent
with endothelial cells and did not undergo fibroblastic transition in
response to FGFs.
Conclusions: Engineered FGF-1s with superior pharmaceutical
properties stimulate corneal endothelial cell growth.
Commercial Relationships: David Eveleth, Trefoil Therapeutics
LLC (P), Trefoil Therapeutics, LLC (E); Xue Xia, None; Ken
Thomas, Trefoil Therapeutics, LLC (I); Michael Blaber, Trefoil
Therapeutics LLC (I), Trefoil Therapeutics LLC (P)
Long-term increase of TGF-β1, TGF-β2 and MCP-1 in the
aqueous humor of pseudophakic Fuchs Dystrophy Eyes
Mario Matthaei, Johannes Gillessen, Philipp S. Muether, Robert
Hoerster, Bjoern O. Bachmann, Arno Hueber, Claus Cursiefen,
Ludwig M. Heindl. Dept. of Ophthalmology, University of Cologne,
Cologne, Germany.
Purpose: To analyze the specific expression of distinct epithelialmesenchymal transition (EMT)-associated proteins in the aqueous
humor of phakic and pseudophakic FECD eyes.
Methods: Fifty aqueous humor samples from phakic FECD eyes
(FECDph), from pseudophakic FECD eyes with more than one year
post-cataract surgery (FECDpsph) and from cataract controls without
FECD (Controlcat) were collected during Descemet membrane
endothelial keratoplasty (DMEK) or cataract surgery. The expression
of EMT-associated proteins (TGF-β1, TGF-β2, TGF-β3, MCP-1,
FGF-basic, TNF-α, Il-1ß) was determined by multiplex bead assay
and comparatively analyzed.
Results: FECDpsph eyes showed significantly elevated expression
compared to FECDph and Controlcat groups respectively for TGF-β1
(p=0.0011 and p<0.0001), TGF-β2 (p=0.0288 and p=0.0076) and
MCP-1 (p=0.0007 and p<0.0001). No differences in EMT-associated
protein levels were detected comparing FECDph eyes and Controlcat
eyes.
Conclusions: Cataract surgery causes long-term elevated levels of
distinct EMT-associated proteins (TGF-β1, TGF-β2, MCP-1) in the
aqueous humor of FECD eyes. This may contribute to accelerated
FECD progression post cataract surgery. No alterations in EMTassociated protein levels were found in phakic FECD eyes.
Commercial Relationships: Mario Matthaei, None; Johannes
Gillessen, None; Philipp S. Muether, None; Robert Hoerster,
None; Bjoern O. Bachmann, None; Arno Hueber, None; Claus
Cursiefen, None; Ludwig M. Heindl, None
RNA toxicity in corneal endothelial tissue in Fuchs dystrophy
Elisabetta Soragni1, JIntang Du1, Ross A. Aleff2, Keith H. Baratz3,
Eric Wieben2, Joel M. Gottesfeld1. 1Cell and Molecular Biology,
The Scripps Research Institute, La Jolla, CA; 2Biochemistry and
Molecular Biology, Mayo Clinic, Rochester, MN; 3Ophthalmology,
Mayo Clinic, Rochester, MN.
Purpose: The strongest genetic association of Fuchs endothelial
corneal dystrophy (FECD) is with an expanded trinucleotide repeat
(CTGCAG) in an intron of the TCF4 gene. The same expanded
repeat sequence in the 3’UTR of the DMPK gene causes Myotonic
Dystrophy type 1 (DM1) via a RNA toxicity mechanism. In DM1,
poly(CUG) transcripts accumulate in foci and sequester the splicing
factor MBNL1, causing missplicing of essential transcripts in skeletal
muscle. Our hypothesis is that the same molecular mechanism causes
RNA toxicity in the corneal endothelium of FECD patients. Because
FECD patients do not present signs or symptoms of DM1, we also
ask whether the molecular signature of RNA toxicity is present in
muscle cells derived from FECD patients.
Methods: Corneal endothelial tissue was obtained at the time of
endothelial keratoplasty, and skin fibroblasts were derived from skin
biopsy specimens from patients with FECD. Using fluorescence in
situ hybridization (FISH), protein-RNA aggregates can be visualized
as RNA foci in the nucleus of affected cells. Immunofluorescence
studies in the same cell types allow for detection of protein
sequestered by the toxic RNA. Induced pluripotent stem (iPS) cells
were also derived from FECD fibroblasts and differentiated to muscle
cells to investigate the presence of RNA foci in this cell type.
Results: Our data show that poly(CUG)-containing transcripts form
RNA foci in fibroblasts derived from FECD patient skin biopsies and
in FECD corneal endothelium and that these RNA foci co-localize
with the splicing factor MBNL1. FISH in muscle cells derived from
DM1 and FECD patients shows that RNA foci are either absent or
less abundant and smaller in size in FECD compared to DM1 muscle
cells.
Conclusions: RNA toxicity may be the cause of FECD, in a manner
similar to DM1. In FECD patients, signs of RNA toxicity are present
in the affected tissue (corneal endothelium) but not (or to a lesser
extent) in iPSC-derived muscle cells, suggesting the possibility that
the expression pattern of TCF4 poly(CUG)-containing transcript
variants determines the pathogenesis of FECD.
Commercial Relationships: Elisabetta Soragni, None; JIntang
Du, None; Ross A. Aleff, None; Keith H. Baratz, None; Eric
Wieben, None; Joel M. Gottesfeld, None
Support: Studies at Scripps were supported by grants from the
California Institute for Regenerative Medicine and the NIH. JD was
supported by a fellowship from the Myotonic Dystrophy Foundation
and by a stem cell fellowship from The Scripps Research Institute.
Studies at Mayo were supported by Research to Prevent Blindness,
Inc.
Development and evaluation of a new laser-assisted experimental
model for Fuchs endothelial corneal dystrophy
Mahdy Ranjbar1, 2, Annekatrin Pahl2, Johannes Rendenbach2, Fred
Reinholz3, Salvatore Grisanti1. 1Dept. of Ophthalmology, University
of Lubeck, Lubeck, Germany; 2Laboratory for Angiogenesis
and Ocular Cell Transplantation, University of Lubeck, Lubeck,
Germany; 3Institute for Biomedical Optics, University of Lubeck,
Lubeck, Germany.
Purpose: Fuchs endothelial corneal dystrophy (FECD) is
characterized by progressive loss of corneal endothelial cells
(CEC), alterations in the extracellular matrix and loss of vision.
Existing disease models focus on the destruction of CEC through the
application of toxic agents or genetic modifications. In this study,
the possibility of an alternative, non-invasive, laser-assisted disease
model for FECD was evaluated.
Methods: For the experiments, freshly enucleated porcine eyes
were positioned in front of a conventional slit lamp combined with
a Nd:YAG-laser. Laser spots were placed into the anterior chamber
with varying energy levels and different distances to the corneal
endothelium. Corneas were excised using a punch trephine and were
then analyzed for laser-mediated lesions by light microscopy and
two-photon microscopy.
Results: Aiming the laser at the anterior chamber results in the
generation of a gas bubble, which spreads out. If the gas bubble
is large enough it can hit the CEC and detach them machanically.
Choosing higher laser energy levels and focusing closely behind the
CEC resulted in larger areas with no CEC. However, in these cases
a ruptured Descemet’s membrane was often observed and the laser
damage even affected the corneal stroma. On the other hand, aiming
the laser too deep into the anterior chamber or selecting lower energy
levels resulted in no detectable laser-mediated effects on CEC.
Nevertheless, it is possible to set a margin for the parameters to create
lesions of CEC-free Descemet’s membrane without rupturing it.
Conclusions: Using a Nd:YAG-laser it is possible to produce small
gas bubbles in the anterior chamber, which damage the corneal
endothelium mechanically, leaving areas of bare Descemet’s
membrane with no CEC attached. This non-invasive, laser-assisted
technique can be used as an alternative experimental model for
diseases with loss of CEC like FECD.
Commercial Relationships: Mahdy Ranjbar, None; Annekatrin
Pahl, None; Johannes Rendenbach, None; Fred Reinholz, None;
Salvatore Grisanti, None
Feasibility of Descemet’s membrane removal during cultivated
corneal endothelial cell injection in rabbit and monkey models
Noriko Koizumi1, Naoki Okumura1, 2, Shinichiro Nakano1, Junji
Kitano1, Shigeru Kinoshita2. 1Biomedical Engineering, Doshisha
University, Kyotanabe City, Japan; 2Department of Ophthalmology,
Kyoto Prefectural University of Medicine, Kyoto, Japan.
Purpose: We developed a cultivated corneal endothelial cell (CEC)
injection treatment for corneal endothelial dysfunction, and have
now initiated clinical research. However, removal of pathologic
Descemet’s membrane (DM) is thought to provide clinical benefits
in cases of Fuchs’ endothelial corneal dystrophy (FECD). Here we
investigated the feasibility of DM removal during cultivated CECinjection therapy in animal corneal endothelial dysfunction models.
Methods: Corneal endothelium was intensively scraped off in 12
eyes of 12 rabbits to create a corneal endothelial dysfunction model.
A 5.0x105 amount of rabbit CECs with rho-associated protein kinase
(ROCK)-inhibitor Y-27632 was then injected into the anterior
chamber of 4 eyes post DM removal (removal group) and into 4 eyes
without DM removal (non-removal group), while 4 eyes without CEC
injection were used as a control. In all eyes, slit-lamp examinations
and corneal thickness- and intraocular pressure (IOP) measurements
were performed for 14 days, followed by immunohistochemical
analysis. As a preliminary experiment, a 5.0x105 amount of cultured
monkey CECs were injected in 1 eye of cynomolgus monkey corneal
endothelial dysfunction model post DM removal.
Results: In the rabbit model, all control eyes failed to recover
corneal clarity, while all eyes in the removal and non-removal
groups recovered corneal clarity at 7-days postoperative. Corneal
thickness was significantly thinner in the non-removal group than
in the removal group at 2-days post-therapy (794.0±29.6μm and
1163.0±32.0μm, respectively; p<0.01), yet at 7-days post-therapy it
had recovered to a similar thickness in both groups (615.3±54.5μm
and 501.3±11.0μm, respectively). No eyes exhibited elevated
IOP. Immunohistochemical analysis showed reconstruction of
a homogeneous monolayer of polygonal cells expressing ZO-1,
N-cadherin, and Na+/K+-ATPase in both the removal and non-removal
groups. The monkey-model cornea became clear at 7-days posttherapy and immunohistochemical analysis showed regeneration of
corneal endothelium expressing function-related proteins.
Conclusions: Our animal corneal endothelial dysfunction model
findings indicate that CEC-injection and DM removal can reconstruct
corneal endothelium. Further studies should provide a clinical
protocol for CEC-injection therapy combined with DM removal to
treat FECD.
Commercial Relationships: Noriko Koizumi, Doshisha University
(P), JCR Pharmaceuticals Co. (P), Senju Pharmaceutical Co. (P);
Naoki Okumura, Doshisha University (P), JCR Pharmaceuticals Co.
(P), Senju Pharmaceutical Co. (P); Shinichiro Nakano, None; Junji
Kitano, None; Shigeru Kinoshita, JCR Pharmaceuticals Co. (P),
Senju Pharmaceutical Co. (P)
Universities from MEXT, Research Center Network for Realization
of Regenerative Medicine from JST
Effect of the cell density of cultivated corneal endothelial cells
on tissue engineering for the treatment of corneal endothelial
dysfunction
Naoki Okumura1, 2, Shinichiro Nakano1, Ayaka Kusakabe1, Ryota
Inoue1, Yugo Okazaki1, Kazuya Kakutani1, Shigeru Kinoshita2, Noriko
Koizumi1. 1Biomedical Engineering, Doshisha University, Kyotanabe,
Japan; 2Ophthalmology, Kyoto Prefectural University of Medicine,
Kyoto, Japan.
Purpose: We have initiated clinical research of a cultured corneal
endothelial cell (CEC) injection therapy for the treatment of corneal
endothelial dysfunction. CEC density (CD) is an important index
for evaluating the health of corneal endothelium in the clinical
setting. Here we investigated the effect of cultivated CEC CD on the
reconstruction of corneal endothelium by cell-injection in an animal
model.
Methods: Corneal endothelium was mechanically removed in 6 eyes
of 6 rabbits. A 5x105 amount of high-CD CECs (1834.7±149.3 cells/
mm2) with Rho-associated protein kinase-inhibitor Y-27632 was
then injected into the anterior chamber of 3 eyes (high-CD group),
while low-CD CECs (503.0±133.7 cells/mm2) were injected into 3
eyes (low-CD group). The anterior segment in each eye was then
evaluated by slit-lamp microscopy for 2 weeks. Corneal thickness and
volume were evaluated by ultrasound pachymetry and a Scheimpflug
camera system (Pentacam® HR). At 2-weeks post treatment, the
phenotype of the reconstructed corneal endothelium was evaluated
by immunohistochemical analysis of ZO-1, N-cadherin, and Na+/
K+-ATPase.
Results: Corneal clarity was recovered in all eyes of both groups.
However, corneal clarity and corneal thickness recovered faster
in the high-CD group than in the low-CD group. Mean corneal
thickness in the high-CD group was significantly lower than that
in the low-CD group at 2-weeks post treatment (391.7±7.0μm and
598.3±11.0μm, respectively). Pentacam® HR examinations revealed
that the mean corneal volume in the high-CD group was significantly
less than that in the low-CD group (42.7±2.2mm3 and 68.4±5.1mm3,
respectively). Immunohistochemical analysis showed regenerated
corneal endothelium in the form of a monolayer that expressed ZO-1,
N-cadherin, and Na+/K+-ATPase. Of interest, the mean regenerated
CEC CD in the high-CD group was 3312.0±429.0 cells/mm2, while
that in the low-CD group was 1642.7±279.2 cells/mm2 (p<0.05).
Conclusions: Our findings suggest that an injection-therapy of highCD cultured CECs can regenerate high-CD corneal endothelium.
Further study is needed to elucidate the optimal criteria of cultured
CEC CD to regenerate high-CD and enable a good prognosis post
corneal endothelial regenerative medicine treatment for corneal
endothelial dysfunction.
Commercial Relationships: Naoki Okumura, JCR Pharmaceuticals
Co.,Ltd. (P), Senju Pharmaceutical Co.,Ltd. (P); Shinichiro Nakano,
None; Ayaka Kusakabe, None; Ryota Inoue, None; Yugo Okazaki,
None; Kazuya Kakutani, None; Shigeru Kinoshita, Alcon (C),
AMO (C), HOYA (C), JCR Pharmaceuticals Co.,Ltd. (P), Otsuka
Pharmaceutical Co. (C), Senju Pharmaceutical Co.,Ltd. (P); Noriko
Koizumi, JCR Pharmaceuticals Co.,Ltd. (P), Senju Pharmaceutical
Co.,Ltd. (P)
Universities from MEXT and Grant-in-Aid for Young Scientists (B)
from JSPS
MicroRNA29b over-expression decreases extra-cellular matrix
protein secretion in Fuchs endothelial corneal dystrophy
endothelial cells
Tetsuya Toyono1, 2, Guadalupe Villarreal1, Laura Kallay1, Tomohiko
Usui2, Shiro Amano3, Albert S. Jun1. 1Wilmer Eye Institute, Johns
Hopkins, Baltimore, MD; 2Ophthalmology, University of Tokyo,
Tokyo, Japan; 3Inoue Eye Hospital, Tokyo, Japan.
Purpose: MicroRNAs are small non-coding RNAs which regulate
gene expression at the post-transcriptional level. We have reported
that the microRNA (miR)-29 family which regulates extracellular
matrix (ECM) expression is decreased in Fuchs endothelial corneal
dystrophy (FECD) patient corneas compared to normal corneas.
Accumulation of ECM proteins in Descemet membrane is an
important pathologic change in FECD. In this study, we transfected
miR29b into immortalized human corneal endothelial cells and
evaluated ECM protein expression levels.
Methods: An immortalized FECD human corneal endothelial cell
line was established by infection of an FECD patient’s corneal
endothelial cells with hTERT lentivirus.MiR29b was transfected into
human FECD corneal endothelial cells using RNAiMAX (Invitrogen)
for 48 hours. Total RNA and protein were extracted. MiR29b
expression change was evaluated with qPCR and ECM proteins
(COL1A1, COL4A1, LAMC1) levels were evaluated with qPCR and
Western blot.
Results: Compared with control, miR29b expression level was
increased to 335.6(±91.0)% and ECM protein expression levels were
decreased. In qPCR results for miR29b transfected cells vs. controls,
COL1A1 was 1.9(±1.4)%, COL4A1 was 7.2(±3.0)% and LAMC1
was 21.5(±7.0)%. In Western blot results for miR29b transfected
cells vs. controls, COL4A1 was 42.5(±25.0)% and LAMC1 was
44.8(±3.1)%.
Conclusions: Over-expression of miR29b decreased ECM protein
expression in FECD human corneal endothelial cells. Thus, miR29
replacement might be a new treatment strategy for FECD aimed
at reducing pathologic production of ECM proteins in Descemet
membrane.
Commercial Relationships: Tetsuya Toyono, None; Guadalupe
Villarreal, None; Laura Kallay, None; Tomohiko Usui, None;
Shiro Amano, None; Albert S. Jun, None
Magnetolipofection - promising tool for targeted gene therapy of
corneal endothelium
Marta Czugala1, 3, Olga Mykhaylyk4, Bernhard B. Singer2, Sebastian
Wesselborg5, Christian Plank4, Friedrich E. Kruse1, Thomas A.
Fuchsluger1, 3. 1Department of Ophthalmology, University Hospital
Erlangen, Erlangen, Germany; 2Institute of Anatomy, University
Hospital Essen, Essen, Germany; 3Department of Ophthalmology,
University Hospital Düsseldorf, Düsseldorf, Germany; 4Institute
of Experimental Oncology, Klinikum rechts der Isar, Technical
University Munich, Munich, Germany; 5Institute of Molecular
Medicine, University Hospital Düsseldorf, Düsseldorf, Germany.
Purpose: Gene therapy of corneal endothelium (CE) faces the
challenge of specific targeting. Topical or intracameral application
does not allow to target the CE precisely. Association of delivery
vectors with magnetic nanoparticles (MNP) can overcome this
problem by delivery to sites of gradient magnetic field application,
thus minimizing the risk of uncontrolled transfection. In this
study, we performed plasmid DNA delivery to CE cells (CEC) in
complexes with MNP and liposomal enhancers upon application
of a gradient magnetic field (magnetolipofection) and evaluated
resulting transfection efficiency, immune compatibility and effect on
endothelial function.
Methods: Transfection efficiency and viability in CEC were assessed
after magnetolipofection with three types of MNP and transfection
enhancers. Expression of CD25 and CD69 activation markers, cell
viability and proliferation in human PBMCs were studied using
flow cytometry. Cell viability was determined by PI or AnnexinV/PI
staining. Transfection efficiency in human corneas was examined by
confocal microscopy.
Results: Magnetolipofection in CEC has proven to be up to four
times more efficient than standard lipofection-based methods. MNP
increased efficiency for XTremeGeneHP by 60% (p=0.0002). Already
with very low dose of pDNA (3pg/cell) and low iron-to-pDNA w/w
ratio (0.25), 39.5±2,7% of cells were successfully transfected while
only 4.6±1.1% of cells were non-viable. For a dose of 6pg/cell,
efficiency was only 30.6±4.0% with 9.8±3.7% of non-viable cells.
In human donor corneas 9.9% transfected cells were observed for a
dose of 360ng pDNA/ml (approx. 2.5pg/CE cell). Biocompatibility
studies with human PBMCs from healthy donors have shown no
significant effects on cell viability or activation of T cells after
magnetolipofection.
Conclusions: In this study, magnetic nanoparticles enabled high
transfection efficiency in CEC at low dose of nucleic acid per cell,
resulting in low toxicity. Our results indicate that the method can
be suitable for in vitro cornea therapy. The data also show that
magnetolipofection itself does not impair endothelial function
or trigger immune response in human PBMCs, thus indicating
good biocompatibility. In addition, the magnetic features of the
nanoparticles enable to control their distribution in vivo. We
conclude, that magnetolipofection provides a promising tool for
targeted gene therapy of corneal endothelial pathologies.
Commercial Relationships: Marta Czugala, None; Olga
Mykhaylyk, None; Bernhard B. Singer, None; Sebastian
Wesselborg, None; Christian Plank, None; Friedrich E. Kruse,
None; Thomas A. Fuchsluger, None
Changes in lipidomic profile of aqueous humor in Fuchs
endothelial corneal dystrophy
Javier Cabrerizo1, J Aritz Urcola1, Marina Calvo de Mora3, Elena
Vecino4, 5, Gerrit Melles2. 1Ophthalmology, University Hospital of
Alava, Vitoria-Gasteiz, Spain; 2Netherlands Institute for Innovative
Ocular Surgery, Rotterdam, Netherlands; 3Hospital Regional
Universitario de Málaga, Málaga, Spain; 4University of the Basque
Country, Bilbao, Spain; 5Grupo de Oftalmo-Biología Experimental
(GOBE), Bilbao, Spain.
Purpose: To identify and determine differences in lipid profiles of
aqueous humor (AH) in patients with Fuchs endothelial dystrophy
(FED).
Methods:
10 pseudophakic patients with FED and 10 phakic controls were
enrolled in our study. Patients with previous history of anterior
segment surgery, anterior segment pathology or intra-ocular
injections were dismissed. Topical ocular medications within the
last 6 months were reported. Aqueous humor was obtained during
the first step of Descemet membrane endothelial keratoplasty
(DMEK) in FED patients and during refractive lensectomy in the
control group. The AH was stored at -40C in the tissue bank for up
to 9 months. Lipidomic ultra performance liquid chromatography
mass spectrometry (UPLC-MS) was used to perform an optimal
profiling of glycerolipids, sterol lipids, sphingolipids and
glycerophospholipids. Metabolite extraction was accomplished
by fractionating the samples into pools of species with similar
physicochemical properties.
Results: The levels of 32 out of 110 lipids change in Fuchs
dystrophy humours when compared to healthy controls. The
concentration of most diacylglycerophosphocholines and 1-ether,
2-acylglycerophosphocholines increase in Fuchs dystrophy humours
when compared to healthy controls. In addition, 9 sphingomyelins
increase significantly in Fuchs humours. Up to 2 long-chain highly
unsaturated cholesteryl esters present higher levels in Fuchs humours
when compared to controls.
Conclusions: The lipid composition of aqueous humor in FED
patients show differences when compared with healthy subjects.
Those changes might be related to pathological changes in the lipid
metabolism of the corneal endothelial cells in FED.
Commercial Relationships: Javier Cabrerizo, None; J Aritz
Urcola, None; Marina Calvo de Mora, None; Elena Vecino, None;
Gerrit Melles, DORC (C)
Protection of corneal endothelial cells by silencing of proapoptotic Bax using nano-particle mediated RNA transfer
Siddharth A. Mahajan1, Marko Pastak2, Olga Rotan3, Matthias
Epple3, Friedrich E. Kruse1, Thomas A. Fuchsluger1. 1Department
of Ophthalmology, University of Erlangen Nurnberg, Erlangen,
Germany; 2Eye Clinic, Tartu University Hospital,, Tartu, Estonia;
3
Institute of Inorganic Chemistry, University of Duisburg Essen,
Essen, Germany.
Purpose: To protect corneal endothelial cells by silencing the
proapoptotic Bax gene using biodegradable calcium phosphate
nanoparticles. We hypothesize that silencing of Bax enhances
survival of corneal endothelial cells under specific apoptotic stimuli.
Methods: Triple shell calcium phosphate nanoparticles with siRNA
against Bax were optimized for human corneal endothelial cell
(EC) transfection using HCEC-12 cell line. Experiments were
performed to obtain the optimum level of transfection using different
concentrations of CaP-siRNA-NPs. The setup showing best balance
between transfection efficiency and cell viability was used for further
experiments. Etoposide was used for induction of apoptosis which
was then analyzed by Annexin V / Propidium Iodide apoptosis assay
using quantitative flow cytometry. Following treatment the cells
were lysed with lysis buffer and lysates were analyzed for levels of
Bax and beta-actin (for normalization) protein expression. Protein
expression was analyzed using ELISA and Western Blotting. Post
transfection viability of cells was assessed by performing MTT cell
viability assay.
Results: Apoptosis was induced in EC using different concentrations
of Etoposide (10, 25, 50, 100 and 250 mg/mL) for specified time
intervals (0, 2, 4, 6, 8, 10 and 12 hours). With etoposide concentration
of 100mg/mL a 3-fold increase in Bax levels was observed after
8hr of apoptosis induction. Transfection of EC with different
concentrations (0.5, 1, 2.5, 5, 10 and 20nM) of CaP-siRNA-NP was
performed. In the presence of the apoptotic stimulus, transfected
EC (with 10nM siRNA) showed decreased level of Bax protein.
Flow cytometry data reveals that EC treated with siRNA show upto
4-fold decrease in apoptotic populations as compared to the untreated
samples.
Conclusions: We demonstrate that silencing of pro-apoptotic Bax
leads to reduced cell death in EC. This strategy could be transferred
to an eye bank setting to protect EC of corneas during storage.
Commercial Relationships: Siddharth A. Mahajan, None; Marko
Pastak, None; Olga Rotan, None; Matthias Epple, None; Friedrich
E. Kruse, None; Thomas A. Fuchsluger, None
Morphological and functional evaluation of mitochondria in a
Fuchs’ endothelial corneal dystrophy cell model
Miu Kitahara1, Naoki Okumura1, 2, Ursula Schlotzer-Schrehardt3,
Friedrich E. Kruse3, Robert D. Young4, Andrew J. Quantock4, Shigeru
Kinoshita2, Noriko Koizumi1. 1Biomedical Engineering, Doshisha
Univertisy, Kyotanabe, Japan; 2Opthalmology, Kyoto Prefectural
University of Mrdicine, Kyoto, Japan; 3Ophthalmology, University
of Erlangen-Nürnberg, Erlangen, Germany; 4Structural Biophysics
Group, School of Optometry and Vision Sciences, Cardiff University,
Cardiff, United Kingdom.
Purpose: The pathogenesis of Fuchs’ endothelial corneal dystrophy
(FECD) has yet to be fully elucidated, though the involvement of
mitochondrial damage and endoplasmic reticulum stress have been
suggested. We recently established an immortalized cellular model of
FECD-patient human corneal endothelial cells (HCECs) (iFECD) and
normal donor-cornea HCECs (iHCED) (article currently in revision).
The purpose of this present study was to investigate the involvement
of mitochondrial dysfunction in FECD by use of these cellular
models.
Methods: To elucidate morphological change of mitochondria,
iFECD and iHCEC were cultured on Transwell® permeable supports
(Corning Inc.) and assessed by transmission electron microscopy
(TEM). Mitochondrial membrane potential (MMP) was evaluated
by fluorescence microscopy following fluorescent probe JC-1
dye staining. The release of cytochrome c from mitochondria to
the cytoplasm was evaluated by Western blotting. To evaluate
the involvement of mitochondria dysfunction in apoptosis, MMP
was evaluated by JC-1 dye, and apoptosis-related proteins such as
caspase 9, caspase 3, and PARP were evaluated by western blotting
in iHCECs stimulated by staurosporine that was used to induce
mitochondrial damage.
Results: TEM showed that mitochondria were dilated in iFECD,
though the morphology of mitochondria was normal in iHCEC.
Staining with JC-1 dye showed that MMP was lower in iFECD than
in iHCEC. Flow cytometry showed that the percentage of depolarized
cells of iFECD was 41.7%, while that of iHCEC was 15.5% (p<0.05).
Western blotting showed a higher level of cytochrome c leakage to
the cytoplasm from mitochondrial fraction in iFECD than in iHCEC.
In addition, western blotting revealed that caspase 9, caspase 3, and
PARP were cleaved after treatment with staurosporine.
Conclusions: The findings of this study indicate that mitochondria
dysfunction is involved in the pathogenesis of FECD, yet further
studies are required to elucidate if mitochondria dysfunction might be
a potent therapeutic target.
Commercial Relationships: Miu Kitahara, None; Naoki
Okumura, Senju phamaceutical (P); Ursula Schlotzer-Schrehardt,
None; Friedrich E. Kruse, None; Robert D. Young, None; Andrew
J. Quantock, None; Shigeru Kinoshita, Senju phamaceutical (P);
Noriko Koizumi, Senju phamaceutical (P)
Analysis of the Barrier Function of Cultured Corneal Endothelial
Cells Isolated from Patients with Fuchs Endothelial Corneal
Dystrophy
Mathieu Theriault1, 2, Olivier Roy1, 2, Isabelle Brunette3, 4, Stephanie
Proulx1, 2. 1Ophtalmologie, Universitï Laval, Quebec, QC, Canada;
2
CUO-Recherche, Centre de recherche du CHU, Quebec, QC,
Canada; 3Ophthalmology Department, University of Montreal,
Montreal, QC, Canada; 4Maisonneuve-Rosemont Hospital Research
Center, Montreal, QC, Canada.
Purpose: The corneal endothelium forms a leaky barrier allowing
the influx and outflux of aqueous humor between the anterior
chamber and the stroma. Fuchs endothelial corneal dystrophy
(FECD) is characterized by a progressive corneal endothelial
decompensation and stromal edema. The goal of this study was to
compare the capacity of FECD and healthy corneal endothelial cells
to form a functional barrier in vitro, in order to gain insight into the
mechanisms underlying the pathology.
Methods: Human corneal endothelial cells (HCEC), isolated from
surgical specimens (FECD; n=6) and age-matched healthy Eye
bank corneas (healthy; n=6) were cultured 24-38 days to obtain a
post-confluent monolayer. Gene profiling analyses were performed
using Agilent SurePrint G3 Human Gene expression microarrays.
Transendothelial resistance (TER) and permeability to 10 kDa
FITC-Dextran were measured in FECD (n=2) and healthy (n=4)
HCEC populations cultured on 12 mm 0.4 mm polycarbonate Isopore
membranes. Measures of membranes without cells were used as
controls. Presence of cadherins (adherens junction marker) and
zonula-occluden-1 (ZO-1; tight junction marker) was also determined
by immunofluorescence.
Results: No significant differences were observed in the
most abundant transcripts of the ZO-1 and cadherin family
members (TJP1: +1.14X, CDH2: -1.05X, CDH11: -1.13X).
Immunofluorescence analysis did not show any difference of
expression between FECD and normal populations for ZO-1 and
pan-cadherin. A lower TER was observed in FECD (22.3 ± 3.5 Ωcm2)
compared to healthy (28.7 ± 8.0 Ωcm2) HCEC populations (p =
0.50). Higher permeability measurements were obtained for FECD
(30.2 ± 5.2% relative to controls) compared to healthy (25.5 ± 3.4%)
populations (p = 0.38).
Conclusions: Results show that within the frame of this study,
cultured FECD and healthy cells had similar junction-related gene
and protein expressions markers. However, the endothelium formed
by FECD cells showed a higher permeability and a reduced TER
compared to healthy cells. Although these early results did not reach
statistical significance because of the small sample size, they suggest
that the ability of FECD cells to form a functional barrier is hindered
in this disease.
Commercial Relationships: Mathieu Theriault, None; Olivier
Roy, None; Isabelle Brunette, None; Stephanie Proulx, None
Support: CIHR, RRSV, ThéCell
The barrier integrity and “pump” function were disrupted by
acute intraocular pressure rise in rat corneal endothelial cells
Xian Li, Jiaoyue Hu, Zhenghao Zhang, Jufeng Meng, Lijun Ye, Zuguo
Liu. Xiamen University, Eye Institute of Xiamen University, Xiamen,
China.
Purpose: The rapid increases in intraocular pressure (IOP) induce
corneal edema in glaucoma patients. We want to investigate the
disruptive effects of acute IOP rise on corneal endothelial cells
(CECs) in rats.
Methods: The Sprague Dawley (SD) rats’ anterior chamber of the
right eyes was irrigated with Ringer’s injection for two hours, and the
left eyes were used as controls. IOPs were measured with handheld
non-contact tonometer once every twenty minutes. At hour 0, hour
48 and day 7 after irrigation, the corneal edema was observed by
silt-lamp microscope, and corneal thickness was measured by Optical
coherence tomography (OCT). At the same time, CECs quantity
and morphology were assessed by noncontact specular microscopy,
and their permeability was determined based on measurements of
FITC-dextran tissue accumulation. Apical junctional complex (AJC)
integrity was evaluated of zonula occludens-1 (ZO-1), vascular
endothelial (VE)-cadherin, filamentous actin (F-actin) and associated
microtubules, as well as Na-K-ATPase by immunofluorescent (IF)
staining and western blot analysis.
Results: The average IOP was 83.3±2.5 mmHg (normal range:
11.1±0.8 mmHg) during anterior irrigation and it decreased to
normal level immediately after irrigation was finished. Instantly
after irrigation, corneal edema became notable, corneal thickness
and the permeability to FITC-dextran significantly increased. The
structures of AJC (ZO-1and VE-cadherin) and cytoskeleton (F-actin
and microtubules) were seriously disrupted by high acute IOP.
The expression of Na-K-ATPase obviously decreased. Then CECs
quantity and morphology (estimated by DAPI and ZO-1 staining
separately) were similar with control eye. Two days after irrigation,
CECs quantity dramatically reduced and they lost regular hexagonal
morphology. However, corneal edema, thickness and permeability
almost recovered to normal level. The structures of AJC and
cytoskeleton, expression of Na-K-ATPase were also restored. Seven
days later, CECs nearly normalized except for decreased cell density.
Conclusions: The structure and function of CSCs were disrupted by
high acute rise of IOP, which resulted in instantly corneal edema and
hereafter the decreased endothelial cells density.
Commercial Relationships: Xian Li, None; Jiaoyue Hu, None;
Zhenghao Zhang, None; Jufeng Meng, None; Lijun Ye, None;
Zuguo Liu, None
Support: National Natural Science Foundation of China:81100638
Elucidating the molecular basis of PPCD3: effect of reduced
ZEB1 levels on corneal endothelial cell migration
Judy L. Chen, Benjamin R. Lin, Ricardo F. Frausto, Andres
Codriansky, Doug Chung, Anthony J. Aldave. Ophthalmology, Jules
Stein Eye Institute, UCLA, Los Angeles, CA.
Purpose: Posterior polymorphous corneal dystrophy 3 (PPCD3)
is characterized by a multilaminar appearance of the typically
unilaminar corneal endothelial cells and is associated with
heterozygous truncating mutations in ZEB1. The purpose of this
study is to determine the effects of reduced ZEB1 protein on human
corneal endothelial cell (HCEnC) migration.
Methods: HCEnC migration was measured using a non-traumatic
wound-healing assay using a HCEnC line (HCEnC-21T). Cells were
seeded in a 12-well plate within two-welled silicone inserts that
established a 500±50 μm gap. Cells were transfected with siRNA
targeting ZEB1 (ZEB1 siRNA) or scrambled siRNA (control siRNA).
As previous experiments demonstrated maximum ZEB1 knockdown
at 48 hours, migration experiments commenced at this time point.
Forty-eight hours post-transfection, the inserts were removed,
and cells were imaged hourly for 15 hours. Migration rates were
calculated using ImageJ software. Data was graphed and statistical
analysis was performed using GraphPad Prism software.
Results: At 15 hours HCEnC transfected with ZEB1 siRNA had
migrated 472 μm (31.4 μm/hr) compared with cells transfected with
control siRNA, which had migrated 471 μm (31.5 μm/hr). Additional
analysis revealed that the early-phase (0-4 hours) migration rate of
ZEB1 siRNA-transfected cells (26.1 μm/hr) was 21% slower than
control siRNA-transfected cells (33.2 μm/hr). Conversely, the latephase (4-16 hours) migration rate of ZEB1 siRNA-transfected cells
was 8% faster (33.3 μm/hr vs. 30.9 μm/hr). For the ZEB1 siRNAtransfected cells, the late-phase migration rate was 23% faster than
the early-phase migration rate.
Conclusions: Although PPCD3 is associated with the appearance of
multilaminar endothelial cells, the role of altered ZEB1 expression
in the development of this abnormality remains to be elucidated.
Although reduced ZEB1 expression does not affect the average
hourly migration rate of HCEnC, two distinct migration phases were
identified in HCEnC with ZEB1 knockdown. An initial reduced
migration rate was followed by an increase in migration rate, the
latter possibly reflecting the induction of a compensatory mechanism
to establish normal migratory properties to endothelial cells in the
setting of reduced ZEB1 levels.
Commercial Relationships: Judy L. Chen, None; Benjamin R.
Lin, None; Ricardo F. Frausto, None; Andres Codriansky, None;
Doug Chung, None; Anthony J. Aldave, None
Support: 1R01 EY022082
OPHTALMIC FEATURES OF SPINOCEREBELLAR ATAXIA
TYPE 7: CORNEAL ENDOTHELIAL CELL DENSITY
CORRELATES WITH ATAXIA RATE AND MUTATION
LENGHT
Lucero Pedro Aguilar5, Aurelio Campos-Romo4, Julio C. Hernandez5,
Victor H. Zuniga-Galvez4, rosalinda diaz4, Aida Jimenez-Corona1,
David Rivera2, Juan Fernandez-Ruiz3, Enrique O. Graue5.
1
Epidemiology, Inst Oftalmologia Conde Valenciana, Mexico,
Mexico; 2Retina, Inst Oftalmologia Conde Valenciana, Mexico,
Mexico; 3Posgrado Neuroetologia, Universidad Veracruzana, Mexico,
Mexico; 4Unidad Periferica Neurociencias, Inst Nacional Neurologia
y Neurociencias, Mexico, Mexico; 5Cornea and Refractive Surgery
Department, Inst Oftalmologia Conde Valenciana, Mexico, Mexico.
Purpose: To analyze the relation between ophthalmologic and motor
changes in spinocerebellar ataxia type 7 (SCA7)
Methods: Design: Case series
Participants: Sixteen SCA7 patients from the State of Veracruz, in
Mexico.
Methods: SCA7 patients underwent a comprehensive ophthalmic
examination including uncorrected and corrected distance visual
acuity, ocular extrinsic motility testing, slit-lamp biomicroscopy
of the anterior segment, fundoscopy and measurement of
intraocular pressure, color vision evaluation with Ishihara color
plates, and optical coherence tomography (OCT) of the optic
nerve and macula. Changes and alterations in the endothelium and
in electroretinographic patterns were explored, and a complete
neurologic evaluation using the Scale for the Assessment and Rating
of Ataxia (SARA) was done as well.
Main outcome measures: optical coherence tomography (OCT)
findings of the optic nerve and macula, alterations in the endothelium
and in electroretinographic patterns, and SARA scores.
Results: All patients showed various degrees of visual impairment
mainly due to macular deterioration. Notably, they also presented
decreased endothelial cell density (ECD).
Pairwise correlations of ECD with number of CAG repeats and
severity of motor symptoms quantified with the SARA scores were
inverse (r=0.46, p=0.083 and r=64, p=0.009, respectively). Further
analyses indicated an average ECD decrease of 48.4 (p=0.006) per
unit of change on the number of CAG repeats, and of 74.6 (p=0.001)
per unit of change on the SARA scores.
Conclusions: The results agree with previous ophthalmological
findings regarding the widespread effect of SCA7 mutation on the
patient’s visual system. However, the results also show a significant
negative correlation of decreased ECD with both CAG repetitions
and SARA scores. This suggests that motor systems degenerate
in parallel with visual systems, although more research is needed
to determine whether the degeneration is caused by the same
mechanisms.
Commercial Relationships: Lucero Pedro Aguilar, None; Aurelio
Campos-Romo, None; Julio C. Hernandez, None; Victor H.
Zuniga-Galvez, None; rosalinda diaz, None; Aida JimenezCorona, None; David Rivera, None; Juan Fernandez-Ruiz, None;
Enrique O. Graue, None

ARVO 2015 Annual Meeting Abstracts 164 Cornea endothelium

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