Download ARVO 2016 Annual Meeting Abstracts 163 Corneal Wound Repair

ARVO 2016 Annual Meeting Abstracts
163 Corneal Wound Repair and Healing
Sunday, May 01, 2016 3:15 PM–5:00 PM
Exhibit/Poster Hall Poster Session
Program #/Board # Range: 1248–1294/D0196–D0242
Organizing Section: Cornea
Program Number: 1248 Poster Board Number: D0196
Presentation Time: 3:15 PM–5:00 PM
A Surgical Cryoprobe for Targeted Transcorneal Freezing to
Treat Corneal Endothelial Dysfunction
Alina Akhbanbetova1, Shinichiro Nakano2, Stacy L. Littlechild1,
Robert D. Young1, Madara Zvirgzdina1, Nigel J. Fullwood3,
Shigeru Kinoshita4, Naoki Okumura2, Noriko Koizumi2,
Andrew J. Quantock1. 1Optometry and Vision Sciences, Cardiff
University, Cardiff, United Kingdom; 2Biomedical Engineering,
Doshisha University, 1-3 Miyakodami-Tatara, Japan; 3Biomedical
and Life Sciences, Lancaster University, Lancaster, United Kingdom;
4
Department of Frontier Medical Science and Technology for
Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto,
Japan.
Purpose: To examine the effects on corneal tissue of localized
freezing induced by a new surgical cryoprobe. The machine was
designed to remove endothelial cells from the posterior surface of the
cornea in a reproducible and targeted manner to aid the treatment of
corneal endothelial dysfunction.
Methods: A freezing console was designed and manufactured based
on the use of nitrous oxide as a cryogen. The console was connected
to one of four different cryoprobes, each with a different freezing
tip (1.8 mm-diameter, flat profile; 2.4 mm-diameter, flat profile; 2.4
mm-diameter concave profile; 3.4 mm-diameter, concave profile) at
which temperatures below -50°C were achieved. In vitro studies were
conducted on 426 porcine corneas, followed by a small number of in
vivo investigations on rabbit corneas. After treatment the epithelial
basement membrane, corneal stroma, and corneal endothelium were
investigated by slit-lamp microscopy, ultrasound pachymetry, and
light and electron microscopy.
Results: In vivo and in vitro the corneal epithelium was destroyed by
freezing, but the epithelial basement membrane remained intact. In
vitro, reproducible corneal endothelial damage was achieved using
the 3.4 mm-diameter cryoprobe tip with the concave profile. The
damage occurred after a short, 3-sec freeze, and was confined to a
circular region of the endothelium located directly under the surface
application position of the cryoprobe tip. Corneal edema was seen in
vivo 24-hrs after freeze injury and was accompanied by alterations to
the arrangement of collagen fibrils, but this resolved by 10-days and
1-month concurrent with endothelial repopulation of the wound area.
Conclusions: Surface corneal freezing using a 3.4 mm-diameter
concave cryoprobe induces transient stromal edema, but leaves the
epithelial basement membrane intact which likely aids epithelial
resurfacing. Localized destruction of the endothelial monolayer was
achieved in a consistent manner, and represents a potentially useful
approach to help treat corneal endothelial dysfunction.
Commercial Relationships: Alina Akhbanbetova, None;
Shinichiro Nakano, None; Stacy L. Littlechild, None;
Robert D. Young, None; Madara Zvirgzdina, None;
Nigel J. Fullwood, None; Shigeru Kinoshita, None;
Naoki Okumura, None; Noriko Koizumi; Andrew J. Quantock,
None
Support: Ser Cymru The Life Sciences Research Network Wales
UK: Research Studenship
Program Number: 1249 Poster Board Number: D0197
Presentation Time: 3:15 PM–5:00 PM
The use of topical insulin to treat refractory neurotrophic corneal
ulcers
Angeline L. Wang, Eric Weinlander, Brandon Metcalf,
David M. Gamm, Michael Struck. Ophthalmology and Visual
Sciences, University of Wisconsin, Madison, WI.
Purpose: Refractory neurotrophic corneal ulcers are clinically
challenging and potentially blinding. Current research focuses on
targeted therapies to improve corneal epithelial healing in these cases.
Insulin has been shown to improve corneal epithelial healing in vitro
and in diabetic animal models; however, clinical experience with
topical insulin in patients with non-healing corneal wounds is limited.
The purpose of this study is to present three cases of refractory
neurotrophic corneal ulcers that were treated with topical insulin.
Methods: Retrospective chart review of patients’ exam findings,
medications, and procedures. Regular insulin at a concentration of
1 unit per mL of artificial tears was prescribed topically as one drop
two or three times daily.
Results: The three patients included a 2-year-old girl with a
history of excised orbital teratoma and proptosis; a 2-year-old boy
with aniridia, congenital glaucoma status post multiple glaucoma
procedures, and bilateral corneal decompensation; and a 24-year-old
woman with a history of herpes zoster keratoconjunctivitis. All three
patients were noted to have decreased or absent corneal sensation in
one eye. Each patient developed a neurotrophic corneal ulcer with
associated epithelial defect and stromal thinning. The ulcers were
refractory to a range of traditional treatments, up to and including
surgical management with permanent tarsorrhaphy in one patient.
The addition of topical insulin resulted in complete corneal reepithelialization ranging from 13 to 25 days following initiation of
treatment. One patient reported increased eye irritation and redness
with the treatment; no systemic side effects were noted.
Conclusions: Topical insulin may be an effective treatment for
refractory neurotrophic corneal ulcers. Proposed mechanisms
include increased corneal epithelial cell migration and restoration
of bidirectional trophic signaling through preservation of corneal
nerves. Insulin has been shown to promote cell migration and closure
of artificial wounds in cultured sheets of corneal epithelial cells. In
diabetic mice, topical insulin appears to slow the loss of sub-basal
plexus corneal nerves. Further study is needed to determine the
clinical efficacy and side effect profile of topical insulin in corneal
wound healing.
Commercial Relationships: Angeline L. Wang, None;
Eric Weinlander, None; Brandon Metcalf, None;
David M. Gamm, None; Michael Struck
Program Number: 1250 Poster Board Number: D0198
Presentation Time: 3:15 PM–5:00 PM
The cornea wound healing properties of retinoic acid passed by
the action of the LOXL4 protein
Loic Blanchon1, Aureli Comptour1, Marion Rouzaire1,
Corinne Belville2, Frederic Chiambaretta1, 4, Vincent Sapin1, 3. 1EA
7281 - Reitnoids Reproduction Developmental Diseases, Université
d’Auvergne, Clermont-ferrand, France; 2GReD - EA7281 - Retinoids,
Reproduction Developmental Diseases, Université d’Auvergne INSERM, Clermont-ferrand, France; 3Biochemistry and Molecular
Biology Department, CHU Clermont-Ferrand, Clermont-ferrand,
France; 4Ophthalmology, CHU Clermont-Ferrand, Clermont-ferrand,
France.
Purpose: Epithelial wound healing is a multistep mechanism
implying a combination of molecular and cellular events.
Following alkali burn traumatisms, cell migration, proliferation and
These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/
to access the versions of record.
ARVO 2016 Annual Meeting Abstracts
differentiation were demonstrated to be required to recover an intact
corneal epithelium and a good visual acuity. Already studied for
its pro-healing properties, vitamin A and its active derivatives (i.e.
retinoic acid / RA) appear to be good candidates to better understand
the cornea wound healing. After having previously demonstrated
in-vitro and in-vivo the effect of RA on wound corneal epithelium
by acting on cell migration, we studied here the lysyl oxidase (LOX)
gene family regulated by RA and the importance of such proteins for
corneal epithelium recovery
Methods: HCE cells were treated by RA for LOX family
members’ characterization and induction by PCR /qPCR and
immunocytochemistry. Promotology experiments were done to study
LOX(s) induction by RA and the RAR (Retinoic Acid Receptor)
isoforms implied. Scratch assays were done after RA treatment
in combination with βAPN (β-aminopropionytrile, a LOX family
enzymatic inhibitor) or after transfection with siRNA against LOX
family members. Cell proliferation and migration were determined
after scratch assay. The right eye of 7 male CD1 per group was
burned with NaOH and treated with RA and w/o βAPN 6 times/
day (d) during 7d. To evaluate wound healing, wounds areas were
quantified from slit lamp photographs at 0 and 7d using imageJ
Results: Among the 5 members of LOX family, only LOXL4
expression (mRNA and protein levels) is increased in HCE treated
with RA from 12 to 60 hours. This regulation passed through the
RARα/RXRα heterodimer fixation on a RARE-DR5 type (Retinoic
Acid Response Element) located in the LOXL4 promoter. In the
in-vitro model, the use of βAPN or siRNA directed against LOXL4
showed a decrease in the wound healing by inhibiting cell migration
promoted by RA treatment. In the in-vivo model the use of βAPN
during RA treatment also inhibits such positive action on wound
healing
Conclusions: We confirmed the importance of RA in cornea
epithelium wound repair. Then, we demonstrated that LOXL4, a gene
implied in the ECM dynamic and induced by RA, is essential for such
phenomenon. This study is one of the first demonstrating a direct link
between vitamin A regulated genes and cornea wound healing and
could constitute a cornerstone of future clinical therapies
Commercial Relationships: Loic Blanchon, None;
Aureli Comptour, None; Marion Rouzaire, None; corinne Belville,
None; frederic chiambaretta, None; Vincent Sapin, None
Program Number: 1251 Poster Board Number: D0199
Presentation Time: 3:15 PM–5:00 PM
Acceleration of EGF expression and related cell behaviors
induced by travoprost were cancelled by further addition of an
EGF receptor inhibitor in corneal epithelium
Yukihisa Takada, Osamu Yamanaka, Takayoshi Sumioka, Yuka Okada,
Shizuya Saika. Wakayama Medical University, Wakayama, Japan.
Purpose: To examined the effects of epidermal growth factor (EGF)
receptor inhibitor, PD168393 (PD), on corneal epithelium treated
with travoprost. We previously reported that travoprost induced the
proliferation of corneal epithelial cells with increased expression of
EGF in vitro and in organ-culture of a mouse cornea (ARVO2015).
Methods: Human corneal epithelial cells (HCE) were cultured
for 24 hrs in the presence or absence of travoprost (0.04 g/l) and/
or PD (10μM). We examined expression of E-cadherin by using
immunohistochemistry and cell proliferation by Alamar blue assay.
Mouse eyeball was organ-cultured for 48h in the presence or absence
of travoprost and/or PD. Expression of EGF, Ki67, phosphorylated
(p) -beta catenin and adhesion molecules (E-cadherin, p-FAK)
and signal pathway related with EGF (pErk, pSmad3) in corneal
epithelium was immunohistochemically examined.
Results: PD blocked travoprost promotion of cell proliferation and
E-cadherin expression in the cultured cells. Promotion of expression
of EGF, Ki 67, p-beta catenin, E-cadherin, p-FAK by travoprost in
corneal epithelium of an organ-cultured mouse eye was counteracted
by further addition of PD. Travoprost induced phosphorylation of Erk
and Smad3, that was also canceled by adding PD.
Conclusions: Acceleration of EGF expression and related cell
behaviors, i.e., promotion of cell proliferation, adhesion molecule
expression, signaling activation, by adding travoprost to corneal
epithelial cell culture or organ-cultured corneal epithelium were
cancelled by further addition of PD. Control of EGF signal could be a
strategy to prevent travoprost-induced corneal epithelial disorder.
Commercial Relationships: Yukihisa Takada; Osamu Yamanaka,
None; Takayoshi Sumioka, None; Yuka Okada, None;
Shizuya Saika, None
Program Number: 1252 Poster Board Number: D0200
Presentation Time: 3:15 PM–5:00 PM
NSAIDs causes corneal epithelial damage by inhibition of
leukotriene B4 receptor 2 signaling
Satoshi Iwamoto1, 2, Tomoaki Koga2, Toshiaki Okuno2,
Akira Murakami1, Akira Matsuda1, Takehiko Yokomizo2.
1
Ophthalmology, Juntendo Univ School of Med, Tokyo, Japan;
2
Biochemistry, Juntendo Univ School of Medicine, Tokyo, Japan.
Purpose: Compromised corneal epithelialization in patients treated
with NSAIDs had been reported. However, pathophysiological
mechanism of delayed corneal wound healing with non-steroidal antiinflammatory drugs (NSAIDs) usage is still not clear. In ARVO 2015,
we reported that a non-canonical BLT2(Leukotriene B4 receptor 2)
ligand, 12(S)-hydroxyheptadecatrienoic acid (12-HHT) accelerated
corneal epithelial wound healing, and 12-HHT production is
inhibited by NSAIDs eye drop. In this study, we further investigated
the mechanism how 12-HHT/BLT2 pathway accelerate corneal
epithelialization.
Methods: Naïve corneal tissue obtained from BLT2(-/-) and congenic
wild type (WT) mice was subjected to next generation sequencing
(NGS) analysis to compare gene expression profiles. Human corneal
epithelial cell line (HCET) overexpressed BLT2 gene was established
and scratch assay experiments were carried out.
Results: The expression of acta2 gene (encoding alpha-smooth
muscle actin) was attenuated in the naïve cornea of BLT(-/-) mouse
compared to WT mouse. In BLT2 overexpressed HCET cells,
accelerated epithelial cell wound closure and increase of acta2 gene
expression was observed.
Conclusions: 12-HHT/BLT2 pathway accelerates corneal epithelial
wound healing with increase of acta2 gene expression.
Commercial Relationships: Satoshi Iwamoto, None;
Tomoaki Koga, None; Toshiaki Okuno, None; Akira Murakami,
None; Akira Matsuda, None; Takehiko Yokomizo
Program Number: 1253 Poster Board Number: D0201
Presentation Time: 3:15 PM–5:00 PM
Effects of loss of TRPV4 function on corneal epithelial wound
healing in mice
Yuka Okada1, Peter S. Reinach3, Masayasu Miyajima2,
Shizuya Saika1. 1Ophthalmology, Wakayama Medical University,
Wakayama, Japan; 2Laboratory Animal Center, Wakayama Medical
University, Wakayama, Japan; 3Wenzhou Medical University,
Wenzhou, China.
Purpose: To determine if transient receptor potential vanilloid 4
(TRPV4), TRPV1-related component, contributes to epithelial wound
healing in a mouse cornea. Growth factor expression pattern was
also evaluated. TRP family consists of groups of TRPV, ankyrin
These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/
to access the versions of record.
ARVO 2016 Annual Meeting Abstracts
1 (TRPA1) or melastatin (TRPM). We previously reported the
involvement of TRPV1 and TRPA1 in corneal epithelial wound
healing in mice (IOVS 2014, ARVO 2015).
Methods: Imunohistochemistry was carried out to examine the
expression opattern of TRPV4 in mouse cornea. Time dependent
closure of a 2.0 mm diameter central corneal epithelial debridement
was monitored in the right eyes of C57BL/6 (wild type, WT, n =61)
and TRPV4-null (KO, n =61) mice for up to 36 h using fluorescein
green staining. BrdU-labeling in WT and KO mice evaluated
proliferative activity during healing. RT-PCR analyzed interleukin
6 (IL-6), substance P (SP) calcitonin gene-related peptide (CGRP),
nerve growth factor (NGF) and transforming growth factor b1
(TGFb1) gene expression levels.
Results: TRPV4 was detected in the basal cells of the corneal
epithelium. The open wound area in the TRPV4 KO mice at 18 and
24 hr was 1.6-fold and 7.34-fold bigger than in the WT counterpart,
respectively. BrdU-labeled cells were fewer in the KO healing
epithelium than in the WT counterpart at 24 and 36 hr (p<0.05).
Increases in CGRP, NGF and TGFb1 gene expression were smaller
than those in the WT counterpart (p<0.05) whereas increases in IL-6
and SP were unaffected by the loss of TRPV4 function.
Conclusions: TRPV4 mediated signaling contributes to increases
in CGRP, NGF and TGFb1 gene expression and repair of a corneal
epithelial debridement in mice.
Commercial Relationships: Yuka Okada; Peter S. Reinach, None;
Masayasu Miyajima, None; Shizuya Saika, None
Program Number: 1254 Poster Board Number: D0202
Presentation Time: 3:15 PM–5:00 PM
Effect of Vitamin D Receptor Knockout on Diabetic Mouse
Corneal Epithelial Wound Healing
Mitchell A. Watsky1, Xiaowen Lu2. 1Cellular Biology & Anatomy,
Augusta University, Augusta, GA; 2Cellular Biology & Anatomy,
Augusta University, Augusta, GA.
Purpose: Diabetes is a significant complicating factor in
numerous pathological conditions, and diabetic keratopathy effects
approximately 70% of all diabetics. Our recent studies determined
that vitamin D receptor (VDR) knockout adversely affects corneal
wound healing. This study was designed to investigate VDR
knockout effects on corneal epithelial wound healing of diabetic
mice.
Methods: The low dose streptozotocin injection method was used
to induce diabetes mellitus in VDR heterozygous (HET) and VDR
knockout (KO) mice along with their wildtype (WT) littermates.
Corneal epithelium wound healing experiments were carried out in
these mice. Mice were anesthetized and 2 mm central wounds were
made using an Alger brush. Wound closure was measured at 0, 12,
20, 28, 36, 48, 60, and 72 h after wounding (measurements stopped
if the wound was completely healed) by photographing fluoresceinstained corneas and digitally outlining the wound margins and
calculating the wound areas using Olympus CellSens Dimension
software. Mice were anesthetized with isoflurane administered
through an anesthesia vaporizer for cornea photography.
Results: Wound areas versus time were plotted and a linear
regression was run on the data. Healing rates were equated to the
regression slope. A rate of -0.018 mm/h was measured in 6-week
diabetic duration KO mice versus a rate of -0.05 mm/h in 6-week
WT diabetic mice. A rate of -0.021 mm/h was measured in 8-week
duration diabetic KO mice versus a rate of -0.046 mm/h in 8-week
WT diabetic mice. KO diabetic mice of 6- and 8-week diabetes
duration had significantly slower healing rates than WT diabetic
mice. There was no significant difference in the healing rate of
4-week diabetic KO mice versus 4-week diabetic WT mice.
Conclusions: KO diabetic mice have significantly slower healing
rates than the WT diabetic mice of 6- and 8-week diabetes duration.
Vitamin D appears to exert a significant influence early in the
development of diabetic keratopathy.
Commercial Relationships: Mitchell A. Watsky, None;
Xiaowen Lu, None
Support: National Institutes of Health (NIH) National Eye Institute
(NEI) Grant R01EY021747
Program Number: 1255 Poster Board Number: D0203
Presentation Time: 3:15 PM–5:00 PM
Role of SKQ1 On Corneal Wound Healing: An In Vitro Model
Using Human Corneal Epithelial Cell Culture Model
Yi Wei1, Penny A. Asbell1, Natalia Perekhvatova2, Maxim Skulachev2,
Anton Petrov2, Boris Chernyak3. 1Ophthalmology, Icahn School of
Medicine at Mount Sinai, New York, NY; 2Mitotech S. A., 42, rue de
la Vallee, Luxembourg; 3Belozersky Institute Of Physico-Chemical
Biology, Moscow State University, Moscow, Russian Federation.
Purpose: SKQ1 (Visomitin) is a novel mitochondrial-targeted antioxidant that holds promise for treatment of ocular surface diseases.
The goal of this study is to determine if SKQ1 plays a role in corneal
wound healing process.
Methods: Human corneal limbus epithelial (HCLE) cell cultures
were pretreated for 1 hour with varying concentrations of SKQ1 (0400 nM). A single-streak wound was then created and fresh medium
containing SKQ1at concentrations corresponding to the amounts for
pretreatment were in place. Photographs were taken at indicated timepoints until wound closure and the resulting images were analyzed
with Image-J. SKQ1 cytotoxicity was determined by a standard
MTT assay. HCLE cell proliferation was evaluated by treating single
cells with varying concentrations of SKQ1 for 6 days and quantified
by MTT assays. HCLE cell migration was evaluated after wounds
treated with SKQ1 and 10 μM pp38 kinase inhibitor SB203580 for
10 hours.
Results: SKQ1 concentrations up to 250 nM showed no toxicity.
Much higher concentrations were needed to produce cytotoxicity.
In comparison to the no-SKQ1 control, addition of 50 nM SKQ1
significantly increased their wound healing rates by 4, 9 and 9%,
corresponding to 4, 8 and 12 hours of SKQ1 treatment. Furthermore,
as low as 25 nM SKQ1 doubled the cell proliferation rates. Finally,
addition of SB203580 completely abolished the stimulated wound
healing by SKQ1.
Conclusions: SKQ1 were shown to significantly enhance the corneal
epithelial wound healing process likely through enhancement of cell
proliferation and migration. The data provide support for SKQ1 as
a promising new therapeutically strategy for treatment of corneal
epithelial wounds and damages.
Commercial Relationships: Yi Wei, None; Penny A. Asbell;
Natalia Perekhvatova, Mitotech, S. A.; Maxim Skulachev,
Mitotech S.A.; Anton Petrov, Mitotech, S. A.; Boris chernyak,
Belozersky Institute Of Physico-Chemical Biology, Moscow State
University, Moscow, Russia (C)
Support: Funded in partial by a research grant from Mitotech
SA Pharmaceuticals, the Martin & Toni Sosnoff Foundation and
Research to Prevent Blindness (RPB)
These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/
to access the versions of record.
ARVO 2016 Annual Meeting Abstracts
Program Number: 1256 Poster Board Number: D0204
Presentation Time: 3:15 PM–5:00 PM
Cell patterning in native and newly formed corneal ECM post
lamellar keratectomy
Pouriska Kivanany1, Byron Weiss2, Elias Choi2, Kevin Lam3,
Nihan Yönet-Tanyeri4, David Schmidtke3, Matthew Petroll1.
1
Department of Ophthalmology, University of Texas Southwestern
Medical Center, Dallas, TX; 2University of Texas Southwestern
Medical School, University of Texas Southwestern Medical Center,
Dallas, TX; 3Department of Bioengineering, University of Texas at
Dallas, Richardson, TX; 4Department of Biomedical Engineering,
Istanbul Medipol University, Istanbul, Turkey.
Purpose: The organized arrangement of the collagen lamellae
plays a key role in corneal transparency. Following injury to the
cornea, quiescent corneal keratocytes transform into fibroblasts and
myofibroblasts. We have previously shown that following freezeinjury, migrating corneal fibroblasts align parallel to the stromal
lamellae. In this study, we compare cell and extracellular matrix
(ECM) patterning within and on top of the stroma following lamellar
keratectomy (LK).
Methods: LK was performed on New Zealand White Rabbits.
From 1 to 60 days after injury, rabbits were monitored using in vivo
confocal microscopy and sacrificed to further investigate cell and
matrix patterning. The corneal tissue was fixed and labeled with
Alexa Fluor 488 phalloidin in situ, and imaged using multiphoton
fluorescence and second harmonic generation (SHG) imaging. Cell
patterning was also assessed in vitro, by plating corneal keratocytes
on top of aligned fibrillar collagen substrates that were generated
using a microfluidics approach.
Results: Immediately following LK, cell death occurred in the native
corneal stroma directly beneath the area of injury. At 7 and 21 days
after LK, confocal and SHG results indicated that the alignment
of fibroblasts repopulating this region was highly correlated with
the orientation of the collagen lamellae. In contrast, cells on top of
the native corneal stroma were randomly arranged, contained more
prominent stress fibers, and secreted fibrotic ECM. Alpha smooth
muscle actin (α-SMA) was also detected in this region, indicating the
presence of myofibroblasts. At 60 days, cells and matrix on top of the
stroma were co-aligned into lamellar-like structures. Corneal haze
peaked at 21 days after LK, and returned to near baseline levels by 60
days. In culture studies, corneal keratocytes stimulated with platelet
derived growth factor aligned parallel to unidirectional collagen
fibers.
Conclusions: Based on these results, we hypothesize that the
topography and alignment of the collagen lamellae direct fibroblast
patterning during repopulation of the native stroma after corneal
injury in the rabbit. Initially, cells on top of the native stroma align
randomly and produce a disorganized ECM, since they do not have
topographic cues to guide them. Over time, cells remodel this fibrotic
ECM to produce a lamellar structure that is similar to the native
corneal stroma.
Commercial Relationships: Pouriska Kivanany, None;
Byron Weiss, None; Elias Choi, None; Kevin Lam, None;
Nihan Yönet-Tanyeri, None; David Schmidtke, None;
Matthew Petroll, None
Support: NIH Grants R01 EY013322 and P30 EY020799, and
Research to Prevent Blindness, Inc.
Program Number: 1257 Poster Board Number: D0205
Presentation Time: 3:15 PM–5:00 PM
Transient and Selective Ingrowth of Lymphatic Vessels into the
Cornea after Incision Injury
Deniz Hos1, Jens Horstmann1, Sebastian E. Siebelmann1,
Franziska Bucher1, Philipp Steven1, Felix Bock1, Reza Dana2,
Claus Cursiefen1. 1Ophthalmology, University of Cologne, Cologne,
Germany; 2Schepens Eye Research Institute, Boston, MA.
Purpose: Corneal lymphangiogenesis contributes to several ocular
pathologies such as dry eye, ocular allergy and corneal graft rejection.
However, a physiological role for corneal lymphangiogenesis,
e.g. during corneal wound healing, has not been described so
far. Therefore, aim of this study was to evaluate whether corneal
lymphangiogenesis occurs during the physiological healing course
after a perforating corneal incision injury.
Methods: A central perforating corneal incision was performed
in C57BL/6 mice. Afterwards, corneal opacity and edema were
scored clinically and analyzed by optical coherence tomography
(OCT). In order to investigate their relationship to corneal hem- and
lymphangiogenesis, blood and lymphatic vessels were analyzed
in whole mounts stained with CD31 and LYVE-1. Real-time PCR
was performed to analyze gene expression of the lymphangiogenic
ligands VEGF-C and VEGF-D and the corresponding receptor
VEGFR-3.
Results: Injured corneas developed opacity and edema, detectable
as an increase of central corneal thickness (CCT), which peaked in
the first two weeks after incision injury (mean CCT in uninjured:
90.1µm; after 1 week: 147.9µm, p<0.01; after 2 weeks: 165.6µm,
p<0.01) and then gradually decreased until corneas became clear
after 4 weeks (mean CCT after 4 weeks: 125.4µm, p>0.05). In
addition, incision injury resulted in selective ingrowth of lymphatic,
but not blood vessels, into the cornea. Corneal lymphangiogenesis
peaked within the first two weeks (mean lymphvascularized area
in uninjured: 2.09%; after 1 week: 7.35%, p<0.001; after 2 weeks:
6.87%, p<0.01) and then regressed (mean lymphvascularized area
after 4 weeks: 3.45%, p>0.05). Furthermore, ingrowth of corneal
lymphatic vessels was accompanied by upregulated gene expression
of VEGF-C (x2.5 after 1 week; x2.0 after 2 weeks; x1.7 after 4
weeks; all p-values <0.01), VEGF-D (x2.0 after 1 week, p<0.01; x1.0
after 2 weeks, p>0.05; x1.1 after 4 weeks, p>0.05) and VEGFR-3
(x14.4 after 1 week, p<0.001; x1.7 after 2 weeks, p<0.05; x3.4 after 4
weeks, p<0.01).
Conclusions: A central perforating incision injury leads to transient
and selective ingrowth of lymphatic vessels into the cornea, which
points to a putative physiological role of lymphangiogenesis during
corneal wound healing.
Commercial Relationships: Deniz Hos, None; Jens Horstmann,
None; Sebastian E. Siebelmann, None; Franziska Bucher, None;
Philipp Steven, None; Felix Bock, None; Reza Dana, None;
Claus Cursiefen, None
Support: German Research Foundation FOR2240 “(Lymph)
angiogenesis and cellular immunity in inflammatory diseases of
the eye” (HO 5556/1-1); EU COST BM1302; Gerok-Program,
University of Cologne
Program Number: 1258 Poster Board Number: D0206
Presentation Time: 3:15 PM–5:00 PM
LncRNA Expression Profile and Bioinformatic Analyses of
LncRNA-gene Network in Corneal Epithelial Wound Healing
Dongsheng Yan, Qiongjie Cao, Xiaoting Zhao, Dewei Peng.
Ophthalmology and Optometry, Wenzhou Medical University,
Wenzhou Zhejiang, China.
These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/
to access the versions of record.
ARVO 2016 Annual Meeting Abstracts
Purpose: Long non-coding RNAs (LncRNAs) are a novel class of
non-protein coding transcripts longer than 200 nucleotides. Recent
studies demonstrated that lncRNAs are essential in a wide range of
physiological and pathological processes. The roles of lncRNAs in
corneal epithelial wound healing, however, remains unknown. In
the present study, we profile their involvement in corneal epithelial
renewal and develop a lncRNA-gene network that affects wound
healing outcome.
Methods: Mouse corneal epithelial cell layers were scratch wounded
and harvested. Total RNA was extracted. We used the Affymetrix
Microarray Platform to analyze differentially expressed lncRNAs
during mouse corneal epithelial wound healing. Real time RTPCR analyses were performed to validate the microarray findings.
Bioinformatic analyses predicted the target genes of candidate
lncRNAs and their involved signaling pathways.
Results: Microarray analysis indicated that 1500 lncRNAs were
significantly downregulated whereas 1253 others were markedly
upregulated during corneal wound healing (fold change>2). The
expression of 8 lncRNAs was successfully confirmed by Realtime
RT-PCR. Bioinformatic analyses revealed involvement of a complex
lncRNA-gene network during corneal epithelial wound healing.
Conclusions: Our results revealed differentially expressed lncRNA
patterns during corneal epithelial wound healing and a complex
lncRNA-gene network that is modulated by these lncRNAs.
Functional analysis will be performed to investigate the roles of
specific lncRNAs in corneal epithelial wound healing.
Commercial Relationships: Dongsheng Yan, None; Qiongjie Cao,
None; Xiaoting Zhao, None; Dewei Peng, None
Support: National Natural Science Foundation of China Grant
81170821, 973 Project (2012CB722303) from the Ministry of
Science and Technology of China, and Science Foundation of
Wenzhou Medical University QTJ11020.
Program Number: 1259 Poster Board Number: D0207
Presentation Time: 3:15 PM–5:00 PM
Copper promotes lysyl oxidase activity in corneal cells and tissues
Santosh K. Muddana1, Hironori Uehara1, Dallas Shi1, Faisal Ahmed1,
Ramesh Rallabandi2, Balamurali K. Ambati1. 1Opthamology, Moran
Eye Center, Salt Lake City, UT; 2Chemistry, University of Utah, Salt
Lake City, UT.
Purpose:
Keratoconus is a corneal disorder that is a leading cause of corneal
transplant, and due to corneal biomechanical weakness secondary
to insufficient collagen cross-linking defects. Mutations in lysyl
oxidase-1 (loxl1), which is responsible to collagen cross linking, have
been identified in keratoconus patients. LOXL1 requires copper as a
co-enzyme. In this study, we examined whether copper treatment can
enhance collagen cross-linking in corneal cells and tissues.
Methods: Human keratoconus corneas were obtained from the
patient undergoing corneal transplantation. Human healthy corneas
were obtained from Utah Lion Eye Bank. Each cornea was bisected
and one half was incubated in 0.0016 mg/ml copper sulfate (CuSo4)
Optisol for 1 week at 25°C and the other half was incubated in
normal optisol.
The Cornea samples were washed with PBS and powdered in liquid
nitrogen and pulverized with a Freezer Mill. Cornea samples were
demineralized with 0.5 M EDTA in 0.05 M Tris buffer, pH 7.4, for 48
h at 4C. The insoluble fraction was washed twice with distilled water
by centrifuging at 5000rpm at 47C for 20 min and lyophilized. The
lyophilized sample was hydrolyzed with 6 N HCl for 24 h at 110°C.
The hydrolysate was neutralized with 2.5 N NaOH and dissolved in
distilled water. Analysis was done for Lysinonorleucine (LNL) and
Histidinohydroxylysinonorleucine (HHL) separately by HPLC.
Corneal stroma cells from normal and keratoconus cornea were
cultured and treated with and without 0.0016mg/mL CuSO4. Then,
the conditioned culture medium was subjected to a peroxidasecoupled fluorometric assay for lox activity assay.
Results: HPLC demonstrated an increase in LNL and HHL in copper
treated keratoconus corneas, while their levels were not detected in
control-treated keratoconus cornea. Loxl1 activity was also increased
with copper treatment in both normal and keratoconus corneal stroma
cells.
Conclusions: CuSO4 treatment increase lysyl oxidase activity and
dramatic increase in LNL and HHL collage crosslinking. This may
indicate a role for copper therapy in keratoconus.
Commercial Relationships: Santosh K. Muddana, None;
Hironori Uehara; Dallas Shi, None; Faisal Ahmed, None;
Ramesh Rallabandi, None; Balamurali K. Ambati, None
Support: EY017950
Program Number: 1260 Poster Board Number: D0208
Presentation Time: 3:15 PM–5:00 PM
The role of blepharitis in the healing course of corneal ulcers
Cynthia I. Tung1, James Reidy2, Ruth M. Mattern2, Sangita P. Patel2, 3.
1
Head and Neck Surgery, MD Anderson Cancer Center, Houston,
TX; 2Ophthalmology, University at Buffalo, Buffalo, NY; 3Research
Service, VA Western New York Healthcare System, Buffalo, NY.
Purpose: Blepharitis is a known risk factor for the development
of infectious keratitis, but the relationship between blepharitis
and the healing course of corneal ulcers has not been defined. We
performed a retrospective clinical study to determine the time to
re-epithelialization of corneal ulcers in the presence and absence of
blepharitis. We hypothesized that the presence of blepharitis would
prolong the time to re-epithelialization of corneal ulcers.
Methods: A retrospective chart review was performed for 23 corneal
ulcer patients that were diagnosed and treated over a 3 and a half year
period (December 2011 to June 2015) by three ophthalmologists at a
university eye institute. Two groups were studied: one with presence
of blepharitis (n=11) and one with absence of blepharitis (n=12) on
clinical slit lamp exam. Inclusion criteria were ICD-9 diagnosis codes
for corneal ulcers (370.0, 370.01, 370.03). Exclusion criteria included
neurotrophic, viral, fungal, acanthamoebic ulcers, ulcers greater than
6mm in longest diameter on presentation, association with uveitis or
endophthalmitis, and history of corneal surgery or significant ocular
surface disease. Number of days were recorded from the first visit
where corneal ulcer was noted to the first visit re-epithelialization
was noted. Student’s t-test and Pearson’s correlation coefficient were
used for statistical analysis.
Results: In the blepharitis group, time to re-epithelialization of the
corneal ulcer was significantly longer (19.3±15.1 days), compared to
the non-blepharitis group (7.3±4.6 days) (p=0.02). Median days to
re-epithelialization were 13 and 6, respectively. In correlating time
to re-epithelialization with age, the non-blepharitis group showed a
weak positive correlation (R = +0.23), while the blepharitis group
showed almost no correlation (R = +0.081).
Conclusions: We conclude that blepharitis may create an
environment of ocular surface inflammation that significantly delays
the healing of corneal ulcers.
Commercial Relationships: Cynthia I. Tung; James Reidy, None;
Ruth M. Mattern, None; Sangita P. Patel, None
Support: RPB unrestricted grant
These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/
to access the versions of record.
ARVO 2016 Annual Meeting Abstracts
Program Number: 1261 Poster Board Number: D0209
Presentation Time: 3:15 PM–5:00 PM
Conjunctival resection for peripheral ulcerative keratitis (PUK)
Ore-oluwa C. Erikitola, Lona Jawaheer, Kanna Ramaesh,
Deepa Anijeet. Tennet’s Institute of Ophthalmology, NHS Greater
Glasgow and Clyde, Glasgow, United Kingdom.
Purpose: Conjunctival resection as management strategy for
PUK, though described 40 years ago, is relatively underutilized.
Conjunctiva adjacent to PUK has been shown to be populated by
inflammatory cells and cytokines. We describe a case series where
conjunctival resection was used early on in the course of the disease,
as a minimally invasive adjuvant treatment.
Methods: This is a retrospective review of 3 eyes of 3 patients with
PUK who had conjunctival resection. Aetiology, clinical presentation,
treatment, clinical course and stability after conjunctival resection
were analysed.
Results: Mean age of two male and one female patient was
68.7 years (range: 60-76 years). All patients had unilateral
disease. Aetiologies included one case each of chronic recurrent
blepharokeratitis (case 1), rheumatoid arthritis (case 2) and ANCA
positive connective tissue disease (case 3). Extent of ulceration was
2 clock hours in case 1 and 6 clock hours in cases 2 and 3. Depth of
thinning was 30 % in case 1 and 80% in cases 2 and 3. Cases 2 and
3 with connective tissue disease had systemic prednisolone in a dose
of 50- 60 mg for less than 2 weeks. All patients had topical steroids
(dexamethasone 0.1%) up to a frequency of 6 times a day for less
than 2 weeks. All patients also received 2 hourly topical lubricants,
as well as anti-metalloproteinase (tetracycline). Patients underwent
excision and recession of adjacent limbal conjunctiva (limbal
conjunctivectomy) as conservative measures failed to make any
significant improvement in clinical course. Conservative measures
were continued post operatively (tapering systemic and topical
steroids, continuing topical lubricants and tetracycline derivative) in
all 3 patients. Ulcerative component healed within 2 weeks. In each
case, surgical treatment was successful in faster healing and tapering
of systemic steroids. No recurrences were reported at 4-12month
follow up.
Conclusions: Conjunctival resection can be an effective and simple
adjuvant therapeutic modality in the treatment of PUK. Removing
adjacent para-limbal source of immune trigger can aid in the
quicker resolution of PUK, with reduced requirement for systemic
immunosuppression with its harmful side effects. Larger comparative
case studies will be needed to confirm this small scale clinical
observation.
Figure 1: Case 2: a) Before and b)16 days after conjunctival resection
Figure 2: a) Before and b) 5 days after conjunctival resection
Commercial Relationships: Ore-oluwa C. Erikitola, None;
Lona Jawaheer; Kanna Ramaesh, None; Deepa Anijeet, None
Program Number: 1262 Poster Board Number: D0210
Presentation Time: 3:15 PM–5:00 PM
Suture Retention Test as a novel Method for Scaffold
Characterization in Ophthalmology
Florian Küng1, 2, Piotr Stafiej1, 2, Dirk W. Schubert2,
Friedrich E. Kruse1, Thomas A. Fuchsluger1. 1Department of
Ophtalmology, Friedrich-Alexander-Universität Erlangen-Nürnberg,
Erlangen, Germany; 2Institute of Polymer Materials, FriedrichAlexander-Universität Erlangen-Nürnberg, Erlangen, Germany.
Purpose: In order to attach a scaffold on a patient sutures are
commonly used. The quantity describing the resistance of a scaffold
against the pull-out of a suture is called ’suture retention strength’.
This quantity is commonly tested for tubular cardial scaffolds. A
novel approach to modify the testing procedure was performed
in order to adjust the suture retention test for characterizing
ophthalmological scaffolds.
Methods: Polycaprolactone (PCL) is dissolved in chloroform and
cast into petri dishes in order to generate films by solution casting.
The amount of PCL was varied in order to cast films of different
thickness. The thickness of each film was measured in a circular
pattern with a gauge. 21 round samples of a diameter of 14 mm were
cut out of each film according to a circular pattern. The samples were
sutured using a Vicryl Polyglactin 910 4-0 suture. The suture was tied
to a loop. The loop was secured with a bolt and more than 50% of the
sample was clamped in a tensile tester. Using a constant deformation
rate, force was applied on the sample until the suture was pulled out.
In order to generate benchmark data from a material currently applied
in ophthalmology 20 samples of amniotic membrane were tested
under the same conditions.
Results: PCL films of 0.2 ± 0.02 g, 0.4 ± 0.02 g, 0.6 ± 0.02 g and
0.8 ± 0.02 g were cast. The thickness of the films was between 7 µm
and 150 µm. Suture retention strength, elongation at the maximum
force and the elongation at rapture were determined for each sample.
The total mean of the PCL data of the suture retention strength is 1.85
± 1.21 N while the mean of the elongation at maximum force is 4.09
± 1.26 mm and the mean of the elongation rapture is 6.75 ± 1.54 mm.
The amniotic membrane with a thickness of 51 ± 8 µm showed a
suture retention strength of 0.20 ± 0.06 N at an elongation of 5.38 ±
1.51 mm and a total elongation of 6.95 ± 1.59 mm.
Conclusions: It is possible to measure samples on the scale of an
ophthalmological scaffold in a suture retention test. The ’suture
retention test’ is a reliable option to characterize the rigidity of
scaffolds used in ophthalmology. By variation of input parameters
first dependencies of the suture retention strength were revealed.
Using the experimental data, a first comparison between the results of
the amniotic membrane and PCL is accessible.
Commercial Relationships: Florian Küng, None; Piotr Stafiej,
None; Dirk W. Schubert, None; Friedrich E. Kruse, None;
Thomas A. Fuchsluger, None
These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/
to access the versions of record.
ARVO 2016 Annual Meeting Abstracts
Program Number: 1263 Poster Board Number: D0211
Presentation Time: 3:15 PM–5:00 PM
Comparative results of cell culture of human corneal epithelial
cells (HCE) and human corneal keratocytes (HCK) on
electrospun nanofiber matrices of Polycaprolactone blended with
Poly(glycerol sebacate) and chitosan
Piotr Stafiej1, 2, Florian Küng1, 2, Daniel Thieme1, Marta Czugala1,
Dirk W. Schubert2, Friedrich E. Kruse1, Thomas A. Fuchsluger1.
1
Department of Ophthalmology, University of Erlangen-Nurnberg,
Erlangen, Germany; 2Institute of Polymer Materials, University of
Erlangen-Nurnberg, Erlangen, Germany.
Purpose: We have previously shown that Polycaprolactone (PCL)/
Poly(glycerol sebacate) (PGS) nanofiber matrices show properties for
ocular surface reconstruction and the possibility to introduce linker
groups for drug attachment. To evaluate the biocompatibility of these
materials, we now evaluated the biological activity of two human
corneal cell types on these matrices.
Methods: The nanofiber matrices were electrospun in different
blends of PCL with PGS and with chitosan. Electrospun fiber meshes
with either random orientation or aligned have been collected on a
substrate and cut into circular samples with a diameter of 14 mm.
After rinsing the samples in PBS buffer overnight and sterilization
by UV-Light the samples were put into 24-well-Plates and incubated
with HCE and HCK cells for 8 days. Wells with PCL foil were used
as controls. During cultivation biological activities were measured
for each sample after 2, after 5 and after 8 days by cell proliferation
assay (WST-8). After the incubation time the samples were prepared
for confocal microscopy and pictures have been taken to check the
cell morphology. (Results shown as random: Mean ± SD / aligned:
Mean ± SD) (AU=Absorbance Units)
Results: HCE and HCK cells could be cultured on each of the
materials, PCL alone, PCL blended with PGS and PCL blended with
chitosan. On aligned fiber meshes an orientation of the HCE and
the HCK cells in direction of the fibers can be detected on confocal
microscopic pictures by checking the growth direction of the F-Actin
fibers. In contrast the cells grew random on the random fiber meshes.
The bioactivity of the HCE cells was lowest on PCL chitosane [0,006
± 0,003 AU / 0,046 ± 0,02 AU] and similar on the fibers with PGS
[0,175 ± 0,12 AU / 0,078 ± 0,03 AU] and PCL alone [0,149 ± 0,07
AU / 0,078 ± 0,01 AU], while the bioactivity for the HCK cells was
low on each material.
Conclusions: Blending PCL with PGS shows good results for
culturing HCE cells, wether further research has to be conducted on
the chitosane material. Combined with the good alignment of the
cells to the oriented fiber meshes the blend of PCL with PGS shows
the best attributes for the further research with the goal to produce
active surfaces for the tissue engineering while further research on
the blend of PCL with chitosane is needed.
Commercial Relationships: Piotr Stafiej, None; Florian Küng;
Daniel Thieme, None; Marta Czugala, None; Dirk W. Schubert,
None; Friedrich E. Kruse, None; Thomas A. Fuchsluger, None
Program Number: 1264 Poster Board Number: D0212
Presentation Time: 3:15 PM–5:00 PM
Soluble Epoxide Hydrolase Inhibition Ameliorates Diabetic
Neurotrophic Keratopathy and Accelerates Delayed Epithelial
Wound Healing in the Diabetic Mouse Corneas
Haijing Sun1, Jiaoyue Hu2, Wei Li2, Fushin X. Yu1, 2. 1Ophthalmology,
Wayne state university, DETROIT, MI; 2Eye Institute, Xiamen
University, Xiamen, China.
Purpose: Soluble epoxide hydrolase (encoded by Ephx2) rapidly
hydrolyzes biologically active epoxyeicosatrienoic acids into the less
biologically active metabolites, dihydroxyeicostrienoic acids and
has been linked to cardiovascular diseases. This study was aimed to
assess the adverse effects of hyperglycemia-induced expression of
EPHX2 in pathogenesis of diabetic keratopathy and its reverse in
EPHX2 deficient mice or by pharmacological inhibition.
Methods: C57BL/6 Ephx2 knockout mice were induced to develop
diabetes by intraperitoneal injection of streptozotocin (STZ). The
expression of EPHX2 detected by cDNA array was verified by
immunohistochemistry. EPHX2 levels and enzymatic activities
were assessed using Western blotting and sEH activity assay. The
normal and diabetic mice were subjected to epithelium debridement
wound and allowed to healing for indicated times. Sensory nerve
regeneration post epithelium wounding was assessed with whole
mount confocal microscopy.
Results: EPHX2 was only expressed in corneal epithelia of diabetic
corneas and its levels increased with the duration of hyperglycemia
in C57BL/6 mice. The levels of EPHX2 and sEH activities inversely
correlated to the rate of epithelial wound closure. Moreover, EPHX2
inhibitor significantly accelerated epithelial wound closure in
diabetic corneas. EPHX2 knockout diabetic mice have increased rate
of epithelial wound closure enhanced delayed nerve regeneration,
similar to the wild-type B6 mice.
Conclusions: Our data showed that EPHX2 expression increases in
diabetic corneas and the elevated EPHX2 plays a detrimental role in
epithelial wound repaired. Targeting EPHX2 pharmacologically may
represent a new approach for treating diabetic keratopathy.
Commercial Relationships: Haijing Sun, None; Jiaoyue Hu, None;
Wei Li, None; Fushin X. Yu, None
Support: NIH grants EY010869 and EY017960
Program Number: 1265 Poster Board Number: D0213
Presentation Time: 3:15 PM–5:00 PM
Human growth hormone released from a biocompatible
hyaluronic acid biomaterial modulates wound healing in an in
vivo corneal chemical burn model
Gina L. Griffith1, Barbara M. Wirostko3, 2, Hee-Kyoung Lee3, 2,
Anthony J. Johnson4, 1, David O. Zamora1. 1Ocular Trauma, United
States Army Institute for Surgical Research, San Antonio, TX;
2
Moran Eye Center, University of Utah, Salt Lake City, UT; 3Jade
Theraputics, Inc., Salt Lake City, UT; 4San Antonio Military Medical
Center, San Antonio, TX.
Purpose: Hyaluronic acid, a ubiquitously expressed polysaccharide,
is of great interest in the bioengineering and regenerative medicine
communities for use as an off-the-shelf biomaterial. In this study,
cross-linked carboxymethylated hyaluronic acid (CMHA-S)-based
film strips were utilized to provide a sustained release of recombinant
human growth hormone (rHGH) and facilitate the repair and
regeneration of damaged ocular tissues. Our purpose was to test the
hypothesis that CMHA-S strips, with and without loaded rHGH, are
biocompatible in vivo and can be securely and safely retained in the
eye for the treatment of corneal epithelial chemical burns.
Methods: The nictitating membranes of 30 New Zealand white
rabbits (~3.0 kg) were removed 3 wks prior to wound creation and
strip placement. Burns 5.5 mm in diameter were created by placing
a circular filter paper soaked in 1N NaOH centrally onto the cornea
for 30 seconds. Wounds were immediately rinsed with sterile
buffered saline, and the eye evaluated using the McDonald-Shadduck
ophthalmic exam and fluorescein staining. Animals were randomly
grouped (n=5 per group) for treatment with control CMHA-S strips
or with CMHA-S strips containing 50 or 150 µg/strip of rHGH. At
one and two weeks post strip placement, eyes were evaluated by
slit lamp and in vivo confocal microscopy. Corneal histology was
performed using H&E and Masson’s Trichrome stain.
These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/
to access the versions of record.
ARVO 2016 Annual Meeting Abstracts
Results: Upon re-hydration, CMHA-S strips exhibited swelling to
yield a clear soft oblong strip of ~4 mm wide by ~15 mm long, able
to be manipulated with forceps and placed into the lower eye cul-desac. All strips were retained in the eye for a minimum of 96 hrs, with
a maximum retention time of 14 days post placement. Wounds treated
with rHGH loaded films exhibited an increase in wound closure
compared to those treated with unloaded strips. Loaded and unloaded
strips were biocompatible and did not reveal any pathological effects
to the eye or surrounding tissues clinically or on histopathology.
Conclusions: CMHA-S film strips are biocompatible and easily
retained in the ocular cul-de-sac. Furthermore, when compared to
unloaded strips, the rHGH-loaded strips are capable of modulating
the re-epithelialization of acute corneal burns. These results advance
the overall efforts to develop the first FDA-approved ocular
pharmaceutical indicated for corneal wound healing.
Commercial Relationships: Gina L. Griffith, None;
Barbara M. Wirostko, Jade Theraputics, Inc. (P), Jade Theraputics,
Inc., Jade Theraputics, Inc. (I), Jade Theraputics, Inc. (F); HeeKyoung Lee; Anthony J. Johnson, None; David O. Zamora, None
Program Number: 1266 Poster Board Number: D0214
Presentation Time: 3:15 PM–5:00 PM
Sphingolipids and TGF-β signaling in the human cornea
Sarah E. Nicholas, Tyler Rowsey, Megan Stiles, Sufiya Khanam,
Nawajes A. Mandal, Dimitrios Karamichos. Ophthalmology,
University of Oklahoma Health Sciences Center, Midwest city, OK.
Purpose: Corneal fibrosis leaves the cornea opaque and can result in
partial or complete vision loss for which the only current treatment
is corneal transplantation. Recently sphingolipids (SPL) have
been linked to corneal fibrosis and found to be modulated by the
transforming growth factor-β (TGF-β) pathway. The aim of this study
is to dissect the role of sphingolipids in corneal fibrosis and their
signaling mechanisms related to the three TGF-β isoforms.
Methods: Healthy human corneal fibroblasts (HCFs) were cultured
in EMEM with 10% FBS and 0.5mM 2-O-α-D-glucopyranosyl-Lascorbic acid in 3D constructs and allowed to grow for 4 weeks in
the presence of 0.1ng/mL TGF-β1, TGF-β2, and TGF-β3. Cultures
without any growth factors served as Controls. After 4 weeks the
cultures were examined for the expression of sphingolipid specific
pathway signaling using Real-Time PCR and Western Blot.
Results: We investigated all five sphingosine-1-phosphate receptors
1 through 5. TGF-β1 and TGF-β3 led to significant upregulation
of sphingosine-1-phosphate receptor 3 (S1PR3) when compared to
Control HCFs. TGF-β2 did not modulate S1PR3. TGF-β2, on the
other hand, led to significant upregulation of sphingosine kinase 1
(SphK1), while TGF-β3 resulted in upregulation of SphK2.
Conclusions: Overall, we have drawn some intriguing signaling
cross talk between sphingolipid molecules and TGF-β isoforms in
human corneal fibroblasts. This study suggests that TGF-β3 driven
upregulation of S1PR3 may represent a novel target in regulating
corneal fibrosis. Future studies will dissect the signaling mechanism
further.
Commercial Relationships: Sarah E. Nicholas, None;
Tyler Rowsey, None; Megan Stiles, None; Sufiya khanam, None;
Nawajes A. Mandal, None; Dimitrios Karamichos, None
Support: EY025256
Program Number: 1267 Poster Board Number: D0215
Presentation Time: 3:15 PM–5:00 PM
Fibroblast-epithelial cell interactions upregulate the expression of
galectin-3 in cornea
Pablo Argueso1, 2, Andrea Cruzat1, 2, Jerome Mauris1, 2. 1Schepens
Eye Research Institute, Massachusetts Eye and Ear, Boston, MA;
2
Department of Ophthalmology, Harvard Medical School, Boston,
MA.
Purpose: Direct intercellular contacts between stromal and
epithelial cells are involved in a number of functions critical to the
development and repair of the cornea. We recently identified a crucial
role for the carbohydrate-binding protein galectin-3 in promoting
matrix metalloproteinase 9 (MMP9) in epithelial cells through its
interaction with the cell surface receptor CD147. The goal of this
study was to determine whether the interaction of fibroblasts with
epithelial cells regulates the expression and function of galectin-3.
Methods: Primary human corneal fibroblasts and immortalized
human corneal-limbal epithelial cells were maintained in DMEM
medium supplemented with 10% serum and keratinocyte serumfree medium, respectively. The presence of galectin-3 and CD147
in whole cell lysates was quantified by Western blot. Affinity
chromatography was performed using a sepharose-conjugated
galectin-3 column. MMP9 secretion was determined by gel
zymography.
Results: Both fibroblasts and epithelial cells produced galectin-3.
Epithelial cells expressed the highly glycosylated forms of CD147,
known to induce MMP production, whereas fibroblasts expressed
glycoforms of lower molecular weight. CD147 from epithelial and
fibroblast origin bound to galectin-3 in a galactose-dependent manner,
however, exogenous galectin-3 failed to induce MMPs in fibroblasts,
in contrast to epithelial cells. Co-culture of fibroblasts with epithelial
cells resulted in an upregulation of galectin-3, concomitant with an
enhanced expression of CD147 and MMP9. The increase in MMP9
was time-dependent, reaching its maximum at 24-48 h, and was
directly related to the number of fibroblasts in culture.
Conclusions: Our results indicate that galectin-3 expression is
dependent on the interaction between epithelial and stromal cells, and
suggest that sustained upregulation of galectin-3 may potentially lead
to stromal matrix degradation and therefore may be clinically relevant
to corneal disease.
Commercial Relationships: Pablo Argueso, None; Andrea Cruzat,
None; Jerome Mauris, None
Support: Supported by NIH/NEI R01EY024031 (PA) and BostonKPro research fund (AC)
Program Number: 1268 Poster Board Number: D0216
Presentation Time: 3:15 PM–5:00 PM
Development of a large animal ex vivo corneal fibrosis model for
translational research
Todd L. Marlo3, Elizabeth A. Giuliano3, Ajay Sharma3, 2,
Rajiv R. Mohan2, 1. 1Veterinary Medicine and Surgery, Biomedical
Sciences, Veterinary Pathology, and Mason Eye Institute, University
of Missouri, Columbia, MO; 2Harry S. Truman Memorial Veterans
Hospital, Columbia, MO; 3Veterinary Medicine & Surgery, University
of Missouri, Columbia, MO.
Purpose: Currently there is no ex vivo model of the equine cornea.
We sought to determine if the equine cornea is suitable as an ex vivo
model. Specifically, to assess the equine cornea’s extracellular matrix
and cellularity after 7 days using two different culture techniques
(an air/liquid interface and immersion system) to determine the best
ex vivo equine corneal model. Our hypothesis is that the air/liquid
interface model would be superior to the immersion model system.
These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/
to access the versions of record.
ARVO 2016 Annual Meeting Abstracts
Methods: Equine corneas with 2 mm of perilimbal sclera (n=14) are
freshly harvested from horses undergoing humane euthanasia and
free of anterior segment disease. One scleral-corneal ring from each
horse is randomly placed in the air/liquid interface organ culture
system with the contralateral scleral-corneal ring being placed in the
immersion condition organ culture system for 7 days. All scleralcorneal rings were evaluated using serial daily gross photography,
histology, RT-PCR and TUNEL assay. Freshly harvested healthy
equine corneas were utilized as controls for all evaluated parameters.
Results: Scleral-corneal rings placed in immersion condition had
complete loss of corneal transparency on gross photography by 7
days, showed a significant level (p<0.05) of stromal disorganization,
significantly increased (p<0.05) αSMA levels on RT-PCR, and
apoptosis on TUNEL assay when compared to controls. The airliquid corneas had weak stromal disorganization on histopathologic
examination and were not significantly different from normal equine
corneal controls on any other evaluated parameter therefore proving
our hypothesis.
Conclusions: The air-liquid culture condition maintained equine
cornea’s cellular extra cellular matrix and preserved corneal
transparency. Conversely, the immersion system resulted in near
complete degradation of the normal equine corneal architecture after
7 days in culture. The air-liquid interface system is a viable option to
maintain a normal equine cornea in an ex-vivo setting for translational
studies.
Commercial Relationships: Todd L. Marlo; Elizabeth A. Giuliano,
None; Ajay Sharma, None; Rajiv R. Mohan, None
Support: Mainly from VAF grant VAF2015-03 (TM); University
of Missouri Phi Zeta grant (TM); Ruth M. Kraeuchi Missouri
Endowment Chair Ophthalmology Fund (RRM); Partially from the
RO1EY17294 National Eye Institute, NIH, Bethesda, Maryland,
USA and 1l01BX00035701 Veterian Health Affairs, Washington DC,
USA grants
Program Number: 1269 Poster Board Number: D0217
Presentation Time: 3:15 PM–5:00 PM
Accelerated healing of corneal endothelial lesions induced by
engineered FGF-1 derivatives
Ralph Bradshaw1, Michael Blaber2, Amuthakannan Subramanian1,
David Ornitz3, David Eveleth1. 1Research, Trefoil Therapeutics, San
Diego, CA; 2Florida State University, Tallahassee, FL; 3Washington
University, St. Louis, MO.
Purpose: Purpose: Engineered versions of FGF-1 are known to be
potent stimulators of dissociated corneal endothelial cell proliferation
in vitro. These experiments tested the ability of eFGF-1s to accelerate
the healing of corneal endothelial lesions in an organ culture model
Methods: Methods: The eFGF-1s TTHX1001 and TTHX1114 was
generated via site-directed mutagenesis and the enhanced stability
and potency evaluated via isothermal equilibrium denaturation and
X-ray crystallography. Stimulation of FGF receptors was evaluated
in BAF3 cells. Mitogenic potency was tested using NIH 3T3 and
primary human corneal endothelial cell cultures. Healing of corneal
endothelial lesions was evaluated in an organ culture system using
trypan blue quantification of the lesion areas.
Results: Results: Both TTHX1001 and TTHX1114 have enhanced
thermodynamic stability relative to the wild type FGF-1 and stimulate
FGF receptors at lower concentrations. Both potently stimulated
the proliferation of human corneal endothelial cells in culture, with
TTHX1114 approximately 100-fold more potent than wild type FGF1. These eFGFs also accelerated the healing of lesions in the corneal
endothelium of rabbit corneas in organ culture. The increased potency
of TTHX1001 and TTHX1114 was not proportional to the degree of
thermodynamic stabilization.
Conclusions: Conclusions: Engineered FGFs can potently accelerate
the healing of corneal endothelial lesions making these agents
potential therapeutics for corneal endothelial dystrophies.
Commercial Relationships: Ralph Bradshaw, Trefoil
Therapeutics; Michael Blaber, Trefoil Therapeutics (C);
Amuthakannan Subramanian, Trefoil Therapeutics; David Ornitz,
Trefoil Therapeutics (C); David Eveleth, Trefoil Therapeutics
Program Number: 1270 Poster Board Number: D0218
Presentation Time: 3:15 PM–5:00 PM
Insight Into the Corneal Wound Healing Response:
Transcriptomic and Metabolomic Analysis of Corneal Epithelial
Cells Challenged by Bacteria
Kimberly Brothers, Stephen Harvey, Nicholas Stella,
Robert M. Shanks. Ophthalmology, University of Pittsburgh,
Pittsburgh, PA.
Purpose: The cornea is an essential transparent barrier that protects
the eye from exposure to microbes on a daily basis. Wound healing
is critical for maintaining this protective barrier. We recently
demonstrated that certain bacteria prevent corneal wound healing in
vitro and ex vivo. With the contact lens-associated keratitis pathogen
Serratia marcescens, LPS was responsible for this inhibition. This
study sought to determine the impact of LPS-containing bacterial
secretomes on the host response of corneal epithelial cells in vitro.
Methods: Growth medium (mock) and sterile bacterial culture
filtrates (secretomes) were added to human corneal limbal epithelial
(HCLE) cells. HCLE supernatants from mock and secretome
treated cells were collected 24 hours post challenge and analyzed
by cytokine array, ELISA, microarray, and metabolomic analysis.
Contents of challenged HCLE cells were also metabolomically
analyzed. To identify the role of TLR4 in S. marcescens inhibition
of wound healing, HCLEs were independently treated with OxPAPC
and CLI-095 inhibitors and tested in cell migration assays.
Results: HCLEs treated with S. marcescens secretomes resulted
in increased IL-1α, IL-1β, GM-CSF, and IL-6 as determined by
cytokine array, ELISA for IL-1 β, and microarray. Metabolomics
from secretome treated HCLEs had elevated levels of
phosphoethanolamine, a phosphorylated amino alcohol associated
with autophagy, lipid signaling, and apoptosis. TLR4 inhibition
of HCLE cells did not prevent S. marcescens secretome inhibition
of corneal wound healing, suggesting this inhibition is TLR4
independent.
Conclusions: Using a combination of transcriptomics and
metabolomics this study provides insight into the impact of bacteria
on human corneal epithelial cells. Together these data support
when HCLEs are treated with S. marcescens secretomes, there is an
increase in pro-inflammatory markers and small molecules consistent
with activation of the autophagy pathway correlating with altering
epithelial cell wound healing.
Commercial Relationships: Kimberly Brothers, None;
Stephen Harvey, None; Nicholas Stella, None; Robert M. Shanks,
None
Support: NH grant F32EY024785
These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/
to access the versions of record.
ARVO 2016 Annual Meeting Abstracts
Program Number: 1271 Poster Board Number: D0219
Presentation Time: 3:15 PM–5:00 PM
Evaluation of a therapeutic anti-TNF-α drug delivery system for
ocular alkali burns
Chengxin Zhou1, 3, Marie-Claude Robert4, 5, Fengyang Lei1, 3,
Vassiliki Kapoulea1, 3, James Chodosh2, 3, Claes H. Dohlman2, 3,
Eleftherios I. Paschalis1, 3. 1Department of Ophthalmology, Schepens
Eye Research Institute-Massachusetts Eye and Ear, Boston, MA;
2
Massachusetts Eye and Ear, Boston, MA; 3Harvard Medical
School, Boston, MA; 4Department of Ophthalmology, Universite de
Montreal, Montreal, QC, Canada; 5Centre Hospitalier de l’Universite
de Montreal, Hopital Notre-Dame, Montreal, QC, Canada.
Purpose: Ocular burns cause corneal inflammation,
neovascularization (CNV) and scarring. We have shown that tumor
necrosis factor alpha (TNF-α) is upregulated after burn and that
prompt TNF-α inhibition improves corneal wound healing. However,
systemic administration of TNF-α inhibitors can cause significant
adverse events, while topical administration is limited by poor
corneal bioavailability and the need for frequent drug application.
This study was designed to test a novel drug delivery system (DDS)
for sustained-release of TNF-α inhibitor to the ocular surface. We
used a rabbit ocular alkali burn model to assess the efficacy of the
DDS in terms of reduction of corneal CNV, opacity and improvement
of corneal wound healing.
Methods: DDS was prepared using porous PDMS/PVA composite
fabrication that was loaded with 80μg infliximab. Evaluation was
performed in 6 Dutch-belted pigmented rabbits that received ocular
alkali burn with 2N NaOH. Three rabbits received subconjunctival
implantation of anti-TNF-α DDS and 3 sham DDS immediately after
the burn. Rabbits were followed with photography for 3 months and
analyzed for CNV, opacity and re-epithelialization. Inflammation was
assessed using CD45 antibody in tissue sections.
Results: Rabbits treated with anti-TNF-α DDS achieved complete
corneal re-epithelialization within 63.6 ± 7.5 days of burn, reduced
CNV (0.13 ± 0.05 % of cornea area) and corneal opacity (central
score = 2.17 ± 0.85; peripheral score = 0.33 ± 0.41 in a 0-4 scale),
while sham DDS treated rabbits exhibited persistent epithelial defect
until the end of the study (92 days), increased CNV (0.31 ± 0.14 %
of cornea area, p<0.05) and corneal opacity (central score = 3.5 ±
0.57, p>0.05; peripheral score = 2.44 ± 1.41, p<0.05). Anti-TNF-α
DDS was well tolerated. The number of CD45+ cells in anti-TNF-α
DDS treated eyes was significantly lower (mean=4322 cells/ cornea)
compared to sham DDS eyes (mean=17049 cells/ cornea, p<0.05).
Infliximab was still present in the DDS 3 months after implantation,
evident by anti-human IgG immunolocalization.
Conclusions: Sustained topical delivery of anti-TNF-α for the
treatment of ocular alkali burn is feasible using the described DDS.
Local anti-TNF-α therapy suppresses corneal inflammation and CNV,
and improves wound healing. The described DDS may be beneficial
to a variety of ocular surface diseases amenable to biologic therapy.
Commercial Relationships: Chengxin Zhou, None; MarieClaude Robert, None; Fengyang Lei; Vassiliki Kapoulea,
None; James Chodosh, None; Claes H. Dohlman, None;
Eleftherios I. Paschalis, None
Support: Boston Keratoprosthesis Research Fund, Massachusetts
Eye and Ear, Boston, MA
Program Number: 1272 Poster Board Number: D0220
Presentation Time: 3:15 PM–5:00 PM
Relaxin 2 augments tissue growth factor β effects on keratocytes
Ulrike Hampel1, 2, Detlev Holland3, Friedrich P. Paulsen1.
1
Department of Anatomy II, Friedrich Alexander University medical
faculty, Erlangen, Germany; 2Ophthalmology, Johannes Gutenberg
University, Mainz, Germany; 3Bellvue Klinik, Kiel, Germany.
Purpose: We previously showed that the pregnancy hormone
relaxin 2 increases corneal epithelial wound healing by influencing
migration and proliferation. To investigate the influence of relaxin 2
on keratocytes and its potential effect on the stroma.
Methods: Primary keratocytes were isolated and cultured from
transplant residuals of donor corneae. Keratocytes were stimulated
with 1 nM relaxin and in combination with 3 ng/ml tissue growth
factor β (TGFβ) for 48 hours. Differentiation of keratocytes to
myofibroblasts was investigated by smooth muscle actin (SMA)
expression with the help of western blot analysis and concomitant
contractility by collagen gel contraction assays. Lumigan, collagens
1A and 1B mRNA expressions were evaluated by real-time RT-PCR
and collagen production was measured in supernatants by sircol
soluble collagen assay.
Results: Relaxin 2 alone does not influence the SMA expression,
but 10 nM relaxin 2 in combination with TGFβ increases the SMA
expression compared to TGFβ alone. Relaxin 2 does not further
increases TGFβ-induced contractility of keratocytes. TGFβ-elevated
collagen 1A expression was reduced by relaxin 2, whereas collagen
1B and lumigan expression was not influenced by relaxin 2. Total
collagen content measured in supernatants was not influenced by
relaxin 2.
Conclusions: Case reports of corneal decompensation during
pregnancy accuse elevated relaxin levels. Our findings do not support
this theory. Relaxin 2 alone does not influence keratocytes. However,
in combination with TGFβ relaxin 2 can increase wound healing
processes of the corneal stroma.
Commercial Relationships: Ulrike Hampel, None;
Detlev Holland, None; Friedrich P. Paulsen, None
Support: DFG HA 6344/2-1
Program Number: 1273 Poster Board Number: D0221
Presentation Time: 3:15 PM–5:00 PM
Targeting alpha v integrins promotes regenerative healing in the
cornea
Stephanie Gillespie, Audrey M. Bernstein. Ophthalmology, Icahn
School of Medicine at Mount Sinai, New York, NY.
Purpose: Scarring and fibrotic disease result from the persistence
of myofibroblasts characterized by high cell-surface expression of
alpha v integrins, which activate the fibrotic factor TGFβ leading to
pathological cell adhesion and fibrotic matrix secretion. We propose
that increasing ubiquitin-mediated intracellular degradation of alpha
v integrins prevents scarring and induces regenerative healing.
Methods: Experiments were performed in supplemented serumfree media on collagen. Transient transfection in primary human
corneal cells (HCFs) with USP10 or control cDNA was performed
using Amaxa Nucleofection. Standard lentiviral infection
using immortalized htert-HCFs was used to generate a USP10
overexpressing cell line. TGFβ activity was measured by coculturing USP10 cells with htert-SMAD-luciferase/GFP reporter
cells and Bright Glo Luciferase Reagent. Detection of a-SMA and
FN-EDA was by RT-PCR, Western blot, and immunocytochemistry.
Porcine corneas, control or wounded by keratectomy, were treated
with control or USP10 siRNA and cultured for 2 weeks prior to
histological examination for fibrotic markers. USP10 deubiqiuintion
of integrin β-chains was examined by immunoprecipitation of
These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/
to access the versions of record.
ARVO 2016 Annual Meeting Abstracts
integrins in Ubiquitin-FLAG overexpressing htert-HCFs and Western
blot for FLAG.
Results: Through genetic screening (RNA seq), we determined
that corneal stromal myofibroblasts overexpressed a subset of
deubiquitinating enzymes (DUBs), which remove ubiquitin
from proteins, saving them from degradation. We found that
overexpression of the DUB, USP10 produced the fibrotic profile:
a) significantly increased αv/β5/β3/β1 protein levels (2.0-3.9 fold),
b) activated TGFβ (70% increase), c) increased FN-EDA and alpha
smooth muscle actin (α-SMA) gene (1.4-2.1 fold) and protein
expression (2.6-5.1 fold), as well as promoting organization of FNEDA fibrils and α-SMA stress fibers. Silencing USP10 in corneal
organ culture prevented induction of fibrotic markers and promoted
regenerative healing in the epithelium and stroma. Mechanistically,
we found that USP10 deubiquitinated integrin β-chains leading to
decreased degradation and increased recycling to the cell surface.
Conclusions: This novel mechanism puts DUB expression at the
head of the cascade that regulates alpha v integrin cell-surface
abundance and fibrosis, and suggests USP10 as a novel anti-fibrotic
target.
USP10 overexpression induces an increase in FN-EDA (2.7 fold)
compared to control.
Commercial Relationships: Stephanie Gillespie, None;
Audrey M. Bernstein, None
Support: This work was supported by The Research to Prevent
Blindness and NIH-NEI R01 EY024942 and T32 GM 062754 (July
2013-July 2015).
Program Number: 1274 Poster Board Number: D0222
Presentation Time: 3:15 PM–5:00 PM
Alterations in corneal nerves in DIO mice may explain changes in
focal adhesion proteins and migration
Vickery E. Trinkaus-Randall, Jenna Meyer, Martin Minns,
Gregory Teicher, Celeste Rich. Biochemistry and Ophthalmology,
Boston University School of Medicine, Boston, MA.
Purpose: The P2X7 purinergic receptor is localized in the corneal
epithelium with a distinct apico-basal polarity. Upon injury, P2X7
expression becomes diffuse at the wound margin and decreases
away from the wound. Inhibitors of the receptor not only attenuate
migration but alter the cytoskeletal rearrangements that are part of
wound healing. DIO mice (pre-diabetic model for Type II diabetes)
exhibit altered levels of the receptor. Our goal was to examine the
corneal nerves in the DIO and control mice and ask if alterations are
associated with compromised cell migration and wound repair.
Methods: Experiments were performed using in vivo, ex vivo organ
culture and in vitro models. DIO and WT mice were sacrificed
at 15 and 7.5 weeks after initiation of respective diets. Corneas
were removed and either fixed or maintained in organ culture after
epithelial debridement to monitor wound closure. A human corneal
epithelial cell line was used to examine cytoskeletal rearrangements
after injury.
Results: Corneas were stained for Tuj1 and P2X7. Nine tiles of the
corneas were imaged separately from the apical surface through the
stromal nerves and stitched into a composite image. Compression
of z-stacks revealed a striking lack of nerves in the DIO mice. In the
control epithelium the nerves were concentrated in the basal region
and only 20% of the nerves were detected in the most apical region.
In the DIO corneas there was a 75% reduction in basal nerves and a
30% reduction in apical nerves. The decrease may explain the lack
of the prototypical whorl detected in control. Analysis of the central
stroma revealed a 2.3 fold greater number of Tuj1 positive nerves in
the DIO that exhibited different morphologies. P2X7 was detected
along the corneal nerves. While P2X7 mRNA is elevated there was
no detectable difference in localization of P2X7 in the unwounded
epithelium. After injury in the DIO corneas the change in P2X7
localization detected previously was absent. Additional experiments
demonstrated that inhibition of P2X7 decreased turnover of focal
adhesion clusters and altered organization of the cytoskeleton at the
leading edge.
Conclusions: The high fat, high calorie diet of DIO mice results in
a lack of corneal sensory nerves and a lack of change in localization
of P2X7 at the wound margin, indicating the importance of P2X7 in
regulating proper early signaling events after corneal epithelial injury.
Commercial Relationships: Vickery E. Trinkaus-Randall, None;
Jenna Meyer, None; Martin Minns, None; Gregory Teicher, None;
Celeste Rich
Support: NIH Grants RO1 EY06000 and R21EY024392, the
Massachusetts Lions FOundation and the New England Corneal
Transplant Fund
Program Number: 1275 Poster Board Number: D0223
Presentation Time: 3:15 PM–5:00 PM
Pathophysiological analysis of corneas 20-years post-Radial
Keratotomy shows a lack of corneal remodelling
I-Ping Loh1, David Lockington2, Charles N. McGhee1,
Trevor Sherwin1. 1Department of Ophthalmology, Faculty of Medical
and Health Science, The University of Auckland, AUCKLAND, New
Zealand; 2University of Glasgow, Glasgow, United Kingdom.
Purpose: Radial Keratotomy (RK) was once a surgical option for
altering refractive power by making radial incisions on the cornea.
However this procedure was often beset with complications and side
effects and is now considered highly controversial. A rare opportunity
enabled the study of the long term pathophysiological changes in a
pair of RK corneas 20 years after the surgery was performed.
Methods: This pair of post-RK corneas was studied histologically
with Masson’s Trichrome and immunohistochemically with
antibodies that targeting the cellular and structural proteins including
integrin α3β1, cytokeratin 3/12 and laminin.
Results: Slit lamp examination and dark field microscopy identified
12 RK incisions in each cornea. Histological examination showed
that the incisions reached a depth of up to 90% of the stroma.
Masson’s Trichrome staining highlighted epithelial plugs at all of
the 12 sites of radial incisions that disrupted Bowman’s membrane
and extended into the anterior stromal region. Immunohistochemical
labelling using integrin α3β1 showed the epithelial plugs were of
basal epithelial origin. New basement membrane material appeared
appeared to be deposited in the epithelial plug forming an acellular
cystic zone inside the plug. The epithelium at the incision sites
appeared thickened and indented.
These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/
to access the versions of record.
ARVO 2016 Annual Meeting Abstracts
Labelling of keratocyte nuclei revealed that fibrotic cells appeared to
have invaded and remained within the plane of the incision. However
a lack of keratocyte migration subsequent to this apparent migration
left a cellular exclusion zone in the stroma which extended ~100
microns either side of the incision. These cellular rearrangements still
persisted 20 years after surgery.
Conclusions: This study demonstrates that cellular rearrangements
following radial keratotomy leaves the stroma with a significant
cellular exclusion zone indicating a lack of corneal remodelling over
a period of 20 years. It also caused the disruption of the Bowman’s
membrane and formation of cellular plugs in the anterior stroma that
are of epithelial origin. These pathological processes may explain
why RK surgery was often beset with complications and this data
could usefully inform corneal healing processes following more
modern procedures such as relaxing incisions.
Commercial Relationships: I-Ping Loh, None; David Lockington,
None; Charles N. McGhee; Trevor Sherwin, None
Support: Auckland Medical Research Foundation Grant 1111010;
Save Sight Society Grant 3622588
Program Number: 1276 Poster Board Number: D0224
Presentation Time: 3:15 PM–5:00 PM
Discovery of FetuinA and global signalling/remodelling modules
driving myofibroblast differentiation during corneal wound
healing in patients
Arkasubhra Ghosh1, 2, Krishnatej Nishtala1, Dhananjay Kumar1,
Rohit Shetty3. 1GROW Research Laboratory, Narayana Nethralaya
Foundation, Bangalore, India; 2Singapore Eye Research Institute,
Singapore, Singapore; 3Cornea and refractive services, Narayana
Nethralaya, Bangalore, India.
Purpose: Stromal cell differentiation is an essential process in
corneal wound healing. Using quantitative proteomics, we attempt to
discover alterations in global protein networks during activation of
quiescent keratocytes to active fibroblasts and myofibroblasts.
Methods: Human corneal fibroblasts(HCF) were transformed into
keratocytes(KT) by culturing in serum free media for 96h. HCF
treated with 1ng/ml TGFβ every 24h for 5 days differentiated
to myofibroblasts(MYO). The cell types were identified by
specific expression of marker genes Aldh3(KT), Thy-1(HCF) and
αSMA(MYO). Cell lysates and patient tear samples were subjected
to quantitative proteomic analysis using iTRAQ labelled tandem LCMS/MS. Tear samples were collected after approval from Institutional
Ethics Committee and written, informed consent from corneal wound
patients(n=6) and age-matched healthy controls(n=6).
Results: Over 700 proteins were identified with 95% confidence
and filtered by >1.5-fold iTRAQ ratio and p<0.05. PANTHER
analysis showed cytoskeletal regulation, protein assembly and
integrin signalling pathway most significantly altered. A novel TGFβ
regulatory protein FetuinA was discovered significantly elevated in
MYO compared to KT suggesting a global role in repair phenotype.
Actin cytoskeleton regulators COF1, PROF were lower in KT
compared to HCF whereas ACTN1, MYL12A, MHC9 were elevated
in MYO. Chaperones GRP78, Endoplasmin, HSP90A/B HSP70,
etc were also increased in MYO. Differentiation and focal adhesion
proteins FN, TLN1, ZYX, FLNA etc were upregulated in HCF and
MYO. These proteins were further validated by immunoblotting.
Proteomic analysis of tears from patients undergoing corneal wound
healing demonstrated elevated FetuinA and cytoskeletal proteins,
recapitulating observations from in vitro model.
Conclusions: We report a unique set of differentiation factors for
wound healing primarily in cytoskeletal and chaperone signalling
modules. We uncover FetuinA as highly elevated in myofibroblasts
and corneal wound patient tears indicating its critical role in
regulating proper healing by modulating TGF-beta function. The data
illustrates distinct interactive protein networks that reveal a specific
response to TGFβ (from wound or exogenous addition) mediated
differentiation process. These novel proteins can serve as drug targets
to treat corneal scarring.
Commercial Relationships: Arkasubhra Ghosh, None;
Krishnatej Nishtala, None; Dhananjay Kumar, None; rohit shetty,
None
Support: Narayana Nethralaya Foundation
Program Number: 1277 Poster Board Number: D0225
Presentation Time: 3:15 PM–5:00 PM
Downstream signal pathway of epidermal growth factor receptor
on the in vitro corneal wound healing model
Masataka Ito1, Yoko Karasawa2, Masaru Takeuchi2. 1Developmental
Anatomy, National Defense Med College, Tokorozawa, Japan;
2
Ophthalmology, National Defense Med College, Tokorozawa, Japan.
Purpose: We have previously reported that, in the in vitro corneal
wound healing model using mouse corneal epithelial cell line,
epidermal growth factor (EGF) promoted re-epithelialization of
denuded area, and that the re-epithelialization did not depend on
cell proliferation. The purpose of this study was to investigate
which downstream signal pathway, MAP kinase pathway or
phosphatidyl inositol 3 (PI3) kinase pathway, is involved in this reepithelialization.
Methods: Corneal epithelial cell line TKE-2 was cultured and
multilayered cultures were scratch-wounded with P10 pipette tip.
Either EGF receptor kinase inhibitor AG1478 (2.5μM), MAP kinase
kinase inhibitor PD08059 (20μM), PI3 kinase inhibitor LY294002
(50μM), or Wortmannin (5μM) was added to the culture and
decrease of denuded area was evaluated. ImageJ software was used
for the measurement of the area. Cell proliferation was evaluated
by BrdU uptake and cell death by colorimetric analyses of lactate
dehydrogenase in proliferating cell cultures.
Results: When EGF receptor kinase inhibitor AG1478 was added,
re-epithelialization was completely inhibited, while inhibition of
re-epithelialization by MAP kinase kinase inhibitor PD08059 was
mild. Similar to AG1478, two PI3 kinase inhibitors LY294002 and
Wortmannin inhibited re-epithelialization completely. Under the
administration of these inhibitors to proliferating cells, BrdU uptake
was suppressed compared with control, while levels of cell death
were not significantly different.
Conclusions: For the re-epithelialization of denuded area, PI3 kinase
pathway is the main pathway of the downstream of EGF receptor.
Commercial Relationships: Masataka Ito, None; Yoko Karasawa,
None; Masaru Takeuchi, None
Support: Grants–in–aid for Scientific Research #26462673
Program Number: 1278 Poster Board Number: D0226
Presentation Time: 3:15 PM–5:00 PM
Intraluminal Microtubule Acetylation Regulates Ocular
Development and Corneal Repair
Lining Cao1, Zhenjie Xu3, 5, Bo Liu2, Ying Yu3, Marcel Alavi1,
Jill Helms2, Matilda F. Chan1, 4, Peter Marinkovich5, Zena Werb3.
1
Department of Ophthalmology, UCSF Medical School, San
Francisco, CA, USA, San Francisco, CA; 2Division of Plastic and
Reconstructive Surgery, Department of Surgery, Stanford School
of Medicine, Stanford, CA, USA, Stanford, CA; 3Department of
Anatomy, UCSF Medical School, San Francisco, CA; 4Francis
I. Proctor Foundation, University of California, San Francisco,
CA; 5Department of Dermatology, Stanford University School of
Medicine, Stanford, CA.
These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/
to access the versions of record.
ARVO 2016 Annual Meeting Abstracts
Purpose: Microtubules (MTs) are dynamic polymers that have
remained the subject of intense investigation for their roles in cell
division, migration and polarity. Acetylated microtubules are present
in virtually all ocular developmental processes but their roles in
normal ocular physiology are unknown. Our study examined the
role of tubulin acetyltransferase, Mec17, in ocular development and
corneal repair.
Methods: Mec17-/- mice were generated by replacing a 9.2 kB
fragment of the Mec17 gene with a LacZ-neomycin reporter. Whole
eyes of littermate WT and Mec17-/- mice at age E14.5 were processed
for H&E staining. Mec17 expression was detected by β-galactosidase
staining and acetylated a-tubulin (Ac-MT) and ZO-1 expression
were analyzed by immunofluorescence staining. Corneal epithelial
and endothelial injuries were induced by mechanical (scratch) and
chemical injury (alkali) respectively.
Results: Mec17-/- mice at E14.5 demonstrate abnormal development
of major ocular structures including the cornea, lens, retina, and optic
nerve with incomplete penetrance. Wild-type adult mice express
Mec17 and in the cornea (all layers), retina (GCL, OPN and IS/OS
layers), and optic nerve. Acetylated alpha-tubulin is expressed in the
retina (GCL, IPL, OPL and IS/OS layers) and optic nerve. Following
corneal epithelial injury, Mec17-/- mice showed delayed epithelial
closure at 8 and 24 hours post-injury compared with littermate WT
mice. Following corneal alkali injury, WT corneal endothelium
displayed regular ZO-1 staining and dot-like acetylated a-Tubulin,
but Mec17-/- corneas displayed disorganized ZO-1 staining and lacked
acetylated a-Tubulin staining.
Conclusions: Mec17 has an important role in normal ocular
development and is highly expressed in the adult cornea, retina, and
optic nerve. Lack of Mec17 expression results in abnormal corneal
epithelial and endothelial repair. These results establish an essential
function of Mec17 in normal ocular developmental and physiologic
processes.
Commercial Relationships: Lining Cao, None; Zhenjie Xu, None;
Bo Liu, None; Ying Yu, None; Marcel Alavi, None; Jill Helms,
None; Matilda F. Chan, None; Peter Marinkovich; Zena Werb,
None
Support: Research Evaluation and Allocation Committee (REAC)
Award
Program Number: 1279 Poster Board Number: D0227
Presentation Time: 3:15 PM–5:00 PM
Effects of Platelet-rich Plasma on Canine Corneal Fibroblasts in
LPS induced Inflammatory Condition
Young Sam Kwon. Department of Veterinary Surgery, Kyungpook
National University, College of Veterinary Medicine, Daegu, Korea
(the Republic of).
Purpose: This experiment was performed to assess the antiinflammatory and proliferative effects of platelet-rich plasma (PRP)
on the canine corneal fibroblasts.
Methods: To examine PRP effects, the inflammation of corneal
fibroblasts was induced by lipopolysaccharide (LPS). The groups
were divided into three groups: control group, DMEM containing
10% fetal bovine serum; LPS group, culture medium with LPStreated group; LPS-PRP group, culture medium with LPS and
5% PRP-treated group. The effect of PRP on the proliferation and
migration was examined in the inflammatory in vitro condition. The
mRNA expressions of the IL-1β, IL-6, IL-10, TNF-α, collagen type
1, MMP-9 and α-SMA were evaluated using RT-PCR.
Results: The 3% or more PRP groups significantly enhanced
proliferation compared to control group in proliferation assay. The
migratory capacity of canine corneal fibroblasts was stimulated with
5% or more PRP group. RT-PCR results showed significant higher
levels of IL-6, collagen type I, MMP-9 and α-SMA compared to LPS
group.
Conclusions: The results of this study suggest that application of
PRP on canine corneal fibroblasts may reduce the LPS-induced
inflammation and enhance corneal stromal matrix remodeling.
Commercial Relationships: Young Sam Kwon
Program Number: 1280 Poster Board Number: D0228
Presentation Time: 3:15 PM–5:00 PM
sd-rxRNA®: Self-Delivering RNAi Compounds Show Potential
for Corneal Indications Following Topical Application
Michael Byrne, Melissa Maxwell, Richard Looby, Katherine Holton,
James Cardia, Lyn Libertine, Pamela A. Pavco, Karen Bulock.
OPHTHALMOLOGY, RXi Pharmaceuticals, Marlborough, MA.
Purpose: sd-rxRNA® are stable oligonucleotides that have features
of RNAi and antisense and result in spontaneous cellular uptake.
When dosed by intravitreal injection sd-rxRNAs are taken up by all
cell layers of the retina within 24 hours and result in dose-dependent,
target-specific mRNA reduction for as long 3 weeks. RXI-109 is an
sd-rxRNA, targeting connective tissue growth factor (CTGF), being
evaluated in a Phase 1/2 clinical trial for subretinal fibrosis associated
with wet AMD. When RXI-109 is dosed by intravitreal injection to
non-human primate, dose-dependent protein reduction is noted in
the retina and the cornea for at least 1 week. Current ocular clinical
focus is to reduce the progression of scarring in the back of the eye.
Scarring is also a major concern for many front of the eye indications,
specifically those involving the cornea.
Here we present new data highlighting topical delivery of sd-rxRNA
to the cornea, administered as eye drops or formulated in a thermoreversible gel, in the presence of an epithelial injury.
Methods: An 8 mm epithelial wound was created on the cornea of
rabbits. sd-rxRNA was dosed topically 4 times a day for 1 day as
eye drops or in a thermo-reversible gel. A second group of animals
with an intact epithelial layer of the cornea were exposed to the same
dosing regimen. At 24 and 48 hours post application, whole eyes
were processed for histological evaluation of cornea cellular uptake
of sd-rxRNA.
Results: Macroscopically, compound was visible in the cornea in the
injured cornea group at 24 hours post dose. The intensity was less at
48 hours. Microscopically, fluorescent sd-rxRNA was visible through
all layers of the cornea in the presence of an epithelial injury at 24
hours when formulated in the thermo-reversible gel, and through
the majority of layers when administered as drops. Cellular delivery
continued to be detectable at 48 hours. No cellular uptake was visible
in the cornea when the epithelial layer was intact.
Conclusions: sd-rxRNA compounds are stable compounds requiring
no additional delivery vehicle to be taken up by cells. RXI-109, a
CTGF-targeting sd-rxRNA being developed to reduce scarring, is
in a Phase 1/2 clinical trial for subretinal fibrosis associated with
late-stage wet AMD. Here we provide evidence of the potential of the
sd-rxRNA platform for development of topical therapeutics for front
of the eye indications.
Commercial Relationships: Michael Byrne, RXi Pharmaceuticals;
Melissa Maxwell, RXi Pharmaceuticals; Richard Looby, RXi
Pharmaceuticals; Katherine Holton, RXi Pharmaceuticals;
James Cardia, RXi Pharmaceuticals; Lyn Libertine, RXi
Pharmaceuticals; Pamela A. Pavco, RXi Pharmaceuticals;
Karen Bulock, RXi Pharmaceuticals
These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/
to access the versions of record.
ARVO 2016 Annual Meeting Abstracts
Program Number: 1281 Poster Board Number: D0229
Presentation Time: 3:15 PM–5:00 PM
Role of Epidermal Growth Factor Receptor and Src-Family
Kinase Activation in Human Corneal Epithelial Wound Healing
Hasan M. Bashir1, Hiroshi Maeno2, Christine Marshall2,
Claire H. Park1, Richu Raju1, John T. Seykora2, Vivian Lee1.
1
Department of Ophthalmology, University of Pennsylvania Perelman
School of Medicine, Philadelphia, PA; 2Department of Dermatology,
University of Pennsylvania Perelman School of Medicine,
Philadelphia, PA.
Purpose: Corneal epithelial wounds are a serious global concern
that can lead to deeper corneal scarring and blindness. Our previous
study suggests activation of epidermal growth factor receptor (EGFR)
and Src-family kinases (SFK) may play a role in corneal epithelial
wound healing in murine derived corneal epithelial cells. To further
understand the role of the EGFR/SFK pathway in wound healing,
wound assays using immortalized human corneal epithelial cells and
primary human corneal epithelial cells were examined in the presence
and absence of tyrosine kinase inhibitors. Using live cell imaging
and Western blot analysis, cellular events were correlated to cell
migration rates.
Methods: Monolayers derived from 2.040 pRSV-T ATCC (2.040)
immortalized cells and primary human corneal epithelial cells
(HCEC) were established and subsequently wounded. Cells were
incubated with small molecule tyrosine kinase inhibitors or vehicle
at various concentrations in pairs. Wound assays were either imaged
with a live cell imaging system and analyzed with Image J software,
or collected as protein lysates 24 hours after wounding for Western
blot analysis.
Results: Cells incubated with tyrosine kinase inhibitors showed
marked inhibition of cell migration into the wound gaps 24 hours
post wounding at all concentrations tested, while cells with vehicle
only demonstrated near complete resolution of the wound area. There
was a strong correlation between cell migration and activation of
EGFR and SFKs. Treatment with tyrosine kinase inhibitors inhibited
cell migration and this correlated with decreased levels of activated
EGFR and SFK in wounded 2.040 and HCEC.
Conclusions: Enhanced corneal epithelial wound healing was
previously observed in murine derived corneal epithelial cells with
increased levels of activated EGFR and SFK. The introduction of
small molecule tyrosine kinase inhibitors inhibited the capacity of
wounded human corneal epithelial cells to migrate and close wounds,
paralleling decreased activation of EGFR and SFK on Western
blot. Results from this study suggest a critical role for EGFR and
SFK activation in human corneal epithelial wound healing with
small molecule tyrosine kinase inhibitors potentiating EGFR/SFK
dependent signaling. Further studies to enhance human corneal
epithelial wound healing through increased activation of EGFR and
SFK should be conducted.
Commercial Relationships: Hasan M. Bashir, None;
Hiroshi Maeno, None; Christine Marshall, None; Claire H. Park,
None; Richu Raju, None; John T. Seykora; Vivian Lee, None
Support: 1K08EY025742-01, K12EY015398, Research to Prevent
Blindness Unrestricted Departmental Grant
Purpose: Determine the correlation between the ultrastructure of the
epithelial basement membrane (EBM) and the presence/absence of
corneal myofibroblasts in scarred corneas after excimer laser surgery,
incisional trauma, and bacterial infection
Methods: Rabbits were divided into 3 groups according to the
mechanism of injury: (1) -9D PRK (VISX Star S4 IR laser); (2)
partial-thickness (~300μm depth) corneal incisions; (3) P. aeruginosa
corneal ulcer (~6x6mm). Fellow eyes were used as unwounded
controls. Immunohistochemical analysis was used to detect the
myofibroblast marker α-SMA and transmission electron microscopy
at 30,000X was performed to determine whether the lamina lucida
(LL) and lamina densa (LD) was present
Results: Group 1 had high numbers of α-SMA+ cells in the anterior
stroma at 1 month after surgery and the EBM was irregular and
lacked typical LL/LD morphology. At 3 months, there were no αSMA+ cells and the EBM was fully regenerated and indistinguishable
from control corneas. Group 2 had opacity at the incisions at 1, 2,
and 3 months after surgery. None of these corneas had α-SMA+ cells
and the EBM overlying the incisions was fully regenerated at all time
points. Group 3 had large numbers of α-SMA+ cells from the anterior
to posterior stroma at 1 month after infection and had no evidence of
LL/LD overlying the corneal opacity (Figure)
Conclusions: Defective EBM correlated with the presence of corneal
myofibroblasts in scarred corneas after high correction PRK or
pseudomonas aeruginosa keratitis, which supports the hypothesis that
generation and persistence of corneal myofibroblasts is regulated by
the EBM after corneal injuries.
Program Number: 1282 Poster Board Number: D0230
Presentation Time: 3:15 PM–5:00 PM
Ultrastructural evidence of defective regeneration of the
epithelial basement membrane as a major factor in the
generation and persistence of corneal myofibroblasts after
corneal injury in rabbits
Gustavo K. Marino, Abirami Santhanam, K P Connie Tam,
Steven E. Wilson. Ophthalmology, Cleveland Clinic, Cleveland, OH.
These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/
to access the versions of record.
ARVO 2016 Annual Meeting Abstracts
Immunohistochemistry for alpha-smooth muscle actin in central
rabbit corneas: (A) Unwounded control; (C) -9D PRK at 1 month
after surgery; (E) Partial-thickness corneal incision at 1 month
after surgery; (G) Cornea at 1 month after a treated pseudomonas
aeruginosa corneal ulcer (400X mag); and Transmission electron
microscopy of the anterior central cornea of rabbits: (B) Unwounded
control; (D) -9D PRK at 1 month after surgery; (F) Partial-thickness
corneal incision at 1 month after surgery; (H) Cornea at 1 month
after a treated pseudomonas aeruginosa corneal ulcer (30,000X mag).
Arrowheads=a-SMA+ cells; arrows=lamina densa; “X”=absence of
lamina lucida/lamina densa.
Commercial Relationships: Gustavo K. Marino, None;
Abirami Santhanam, None; K P Connie Tam; Steven E. Wilson,
None
Support: Supported by EY10056, EY015638 and Research to
Prevent Blindness, New York, NY
Program Number: 1283 Poster Board Number: D0231
Presentation Time: 3:15 PM–5:00 PM
Desmin is a Novel Druggable Regulator of Corneal Fibrosis
Alexandra Pietraszkiewicz, Christopher Hampton, Ling Lei,
Paola Bargagna-Mohan, Royce Mohan. Neuroscience, University of
Connecticut Health Center, Southington, CT.
Purpose: The intermediate filaments (IFs) vimentin and desmin are
coordinately upregulated in myofibroblasts during corneal fibrosis
and their downregulation by the potent IF-targeting drug withaferin
A (WFA) is protective against fibrosis and haze (JBC 2012, 287: 989-
1006). In this study, we have employed mice deficient for desmin to
unravel how desmin contributes to corneal fibrosis.
Methods: Wild-type (WT) 129Svev litter mates, desmin hemizygous
(Des+/-) and homozygous (Des-/-) knockout mice were employed.
Mice were anesthetized by intraperitoneal injection of ketamine and
xylazine, and corneas were anesthetized with proparacain eye drop.
A 1 μl drop of dilute 0.15 M NaOH was applied for 1 minute, the
cornea was washed with saline solution, and the corneal and limbal
epithelium was removed by scraping with a blunt Tooke corneal
knife. After 7, 14 or 30 days of recovery, mouse corneas were
assessed for corneal opacity and mice sacrificed. The enucleated
eyes were either cryosectioned for immunohistochemistry (IHC) or
corneas dissected and subjected to western blot (WB) analysis. In
other studies, injured Des-/- mice were also treated with vehicle or 2
mg/kg/d WFA for 14 days.
Results: We discovered that the injured corneas of Des-/- mice like
those of WT mice lose corneal transparency and become fibrotic.
Surprisingly, Des+/- corneas were protected against corneal fibrosis
and revealed absence of a-smooth muscle actin and vimentin
upregulation in repairing stromal cells. Recently, we showed that
vimentin becomes phosphorylated at serine 38 residue (pSer38Vim)
and the expression of this biomarker is correlated with corneal
fibrosis (PlosOne 2015, 10(7):e0133399). Therefore, we investigated
the expression of pSer38Vim in corneas of desmin-deficient mice.
We discovered that the healing Des+/- corneas, unlike fibrotic Des/or WT corneas, show very low expression levels of pSer38Vim.
The expression of pSer38vim was also altered in Des-/- corneas by
treatment with WFA.
Conclusions: Vimentin and desmin play overlapping roles in wound
repair and fibrosis, however, depending of gene dosage, the degree of
desmin loss can have protective or pathological consequences. Our
studies, for the first time, illuminates that the phosphorylation status
of vimentin is also governed by desmin during wound repair, and that
targeting of pSer38vim by WFA in desmin deficiency can prevent
corneal fibrosis.
Commercial Relationships: Alexandra Pietraszkiewicz,
None; Christopher Hampton, None; Ling Lei, None;
Paola Bargagna-Mohan; Royce Mohan, University of Kentucky
Research Foundation (P)
Support: NIH Grant EY016782
Program Number: 1284 Poster Board Number: D0232
Presentation Time: 3:15 PM–5:00 PM
A Novel Vimentin-Targeting Probe for Imaging Ocular Fibrosis
Royce Mohan1, Santosh Keshipeddy2, Dennis Wright2,
Paola Bargagna-Mohan1. 1Neuroscience, University of Connecticut
Health Center, Farmington, CT; 2Pharmaceutical Sciences, University
of Connecticut, Storrs, CT.
Purpose: Vimentin is a binding target for the small molecule
withaferin A (WFA). Here we have synthesized a novel WFA
fluorescent analog to investigate the dynamic motility of vimentin
filaments in cell cultures and fibrotic expression of vimentin in vivo.
Methods: WFA was synthetically appended with a fluorescent tag
to develop the imaging probe WFA-BODIPY-FL. In cell labeling
assays, WFA-BODIPY-FL was added for 1 h, washed and cultured
for 18 h. Cells were fixed and counter stained with antibody against
serine 38 phosphorylated vimentin (pSer38vim). In cell spreading
assays, rabbit corneal fibroblasts were trypsinized and plated on cover
slips. After 1 h, WFA-BODIPY-FL (100 nM to 1 μM) was added to
the cells for 5 min in serum-free medium, cells washed and allowed
to spread. Serial images were collected every 5 seconds for 10 mins.
For in vivo studies, 129Svev mice were subjected to the alkali injury
model (JBC 2012, 287:989-1006) to promote corneal fibrosis. At
These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/
to access the versions of record.
ARVO 2016 Annual Meeting Abstracts
14 days post injury mice were injected with a single intraperitoneal
injection of WFA-BODIPY-FL and sacrificed after 1 h. Cryosections
from mouse eyes were fixed and stained with antibody against
vimentin.
Results: We demonstrate that WFA-BODIPY-FL labels vimentin
intracellularly efficiently at 250 nM. Cells labeled for 18 h showed
WFA-BODIPY-FL fluorescence overlapped with antibody staining
for pSer38vim. In cell spreading assays, depolymerized soluble
vimentin shows an abundance of fluorescence around the perinuclear
region. Short squiggle- and dot-like vimentin structures showed
bidirectional movement and increasingly extended towards the cell
periphery. Some dot-like structures moved at rapid speeds of 0.5
micrometers/sec corroborating data from published transfection
studies that used hybrid vimentin-green fluorescent protein
expression. Finally, in injured mice injected with WFA-BODIPY-FL
we found intense fluorescence in corneal myofibroblasts that costained for vimentin. Normal uninjured mice injected with WFABODIPY-FL did not show this intense green fluorescence in the
corneal keratocytes.
Conclusions: We have developed a first-in-class in vivo imaging
probe for studying the dynamic organization of vimentin filaments.
WFA-BODIPY-FL is also bioavailable in mice and shows remarkable
localization of myofibroblasts in fibrotic corneas. This biomarkerimaging reagent will be very useful as a diagnostic tool in ophthalmic
applications for disease and injury-related fibrosis.
Commercial Relationships: Royce Mohan; Santosh Keshipeddy,
None; Dennis Wright, None; Paola Bargagna-Mohan, University
of Kentucky Research Foundation (P)
Support: NIH Grant EY016782
Program Number: 1285 Poster Board Number: D0233
Presentation Time: 3:15 PM–5:00 PM
Differential Effects of Topical Mitomycin C and Steroid on
Wound Healing and Optics after Photorefractive Keratectomy in
Cats
Holly B. Hindman, Margaret DeMagistris, Christine Callan,
Thurma McDaniel, Krystel R. Huxlin. Ophthalmology, Univ of
Rochester, Rochester, NY.
Purpose: To assess the differential effects of topically applied agents
on myofibroblast transformation and optical outcomes following
photorefractive keratectomy (PRK) in a cat model.
Methods: 26 eyes of 13 cats underwent -10 D myopic PRK. Cats
were then assigned to 1 of 3 treatment groups: control, Mitomycin
C (MMC – 0.02% for 2 mins) and topical steroid (BID for 2 wks).
Corneal thickness and reflectivity were measured using optical
coherence tomography (OCT) and wavefront aberrations were
quantified using a Hartmann-Shack aberrometer. Cats were sacrificed
for histology at 2, 4, and 12 wks post-PRK. Corneas were excised,
fixed and stained immunohistochemically for alpha-smooth muscle
actin (α-SMA).
Results: 2 weeks after PRK, significant differences were noted with
respect to stromal (KW=9.576, P<0.01) and epithelial thickness
changes (KS=7917, P<0.05) between groups. Control and steroidtreated eyes had increased stromal thickness and decreased epithelial
thickness relative to MMC-treated eyes. MMC treatment also resulted
in a thinner band of α-SMA staining and lower stromal reflectivity
2 wks post-operatively than in control and steroid-treated eyes. By
4 wks post-PRK, there was no detectable α-SMA in MMC-treated
eyes, whereas control and steroid-treated eyes continued to express
it. By 12 wks post-PRK, stromal reflectivity in MMC-treated eyes
(but not control or steroid-treated eyes) returned back to baseline.
By 12 wks post-PRK, there were also significant differences in the
amount of defocus change induced by PRK between the 3 groups
(KW=9.269, P<0.0005, D=7.75, P< 0.01), with MMC eyes exhibiting
the greatest treatment effect (defocus change: -5.45 ± 0.17µm relative
to baseline). Steroid-treated eyes showed moderate regression
(defocus change: -3.30 ± 0.35 µm), while control eyes had the most
severe regression (defocus change: -2.04±0.36 µm). However, by
12 wks, no significant differences in induced higher order root mean
square (HORMS) existed between groups (control: +0.39 ± 0.34 µm,
steroid: +0.60 ± 0.15 µm, MMC: +0.91 ± 0.17 µm; KW = 2.35,
P=0.34).
Conclusions: Relative to steroid-treated and control eyes, MMC
treatment was associated with more favorable wound healing
responses and optical outcomes after PRK. This included a decrease
in the amount and length of α-SMA expression, greater epithelial
stability, and less myopic regression.
Commercial Relationships: Holly B. Hindman, None;
Margaret DeMagistris, None; Christine Callan, None;
Thurma McDaniel, None; Krystel R. Huxlin, None
Support: NIH 2R01EY015836-11, Research to Prevent Blindness
Program Number: 1286 Poster Board Number: D0234
Presentation Time: 3:15 PM–5:00 PM
Characterization of a quantitative model of corneal
inflammation, wound healing, and fibrosis in the rabbit
David Culp1, Elizabeth Schaefer2, Maria-Grazia Spiga1,
Aliah Hackney1, Nicholas Smith1, Brian C. Gilger2. 1Powered
Research, RTP, NC; 2Clinical Sciences, North Carolina State
University, Raleigh, NC.
Purpose: To develop and characterize a sensitive and translatable
model of corneal wound healing and fibrosis in the New Zealand
White Rabbit.
Methods: An 8 mm central corneal defect of approximately 10-15%
stromal depth was created using a corneal trephine and lamellar
resection. Dexamethasone (DEX) or balanced salt saline (BSS) were
administered topically 4 times a day for 28 days after keratectomy.
Hackett-McDonald (HM) ocular inflammatory scores and area of
fluorescein positive staining to evaluate re-epithelialization were
evaluated every 12 hours for 5 days, then weekly through 28 days.
Stromal fibrosis, haze, and corneal thickness were measured by SDOCT (Envisu, Bioptigen) and histopathology at 12, 24 and 48 hours
and on days 7 and 28.
Results: Cumulative HM scores were significantly lower in eyes
treated with DEX compared to BSS for the first 4 days after surgery;
however, area of fluorescein retention (re-epithelialization of the
wound) was higher in DEX compared to BSS-treated eyes through 7
days and time to complete re-epithelization was longer by 3 days in
DEX vs BSS eyes. On SD-OCT, corneal opacity scores on day 7 were
similar between DEX and BSS treated eyes, but by day 28 there was
a 60% reduction in stromal fibrosis in DEX treated eyes. Histologic
scores in the anterior segment and cornea were consistently lower in
DEX treated eyes on both 7 and 28 days after keratectomy.
Conclusions: Using this corneal wound healing model, we
demonstrated that topical DEX inhibited ocular inflammation, corneal
fibrosis, and opacity compared to BSS. This keratectomy model
allows quantitative assessment of specific anti-inflammatory and
wound healing therapies, and can be used in pre-clinical development
to provide effective proof of concept of novel compounds or delivery
methods.
Commercial Relationships: David Culp, Powered Research;
Elizabeth Schaefer, Powered Research (C); Maria-Grazia Spiga,
Powered Reserach; Aliah Hackney, Powered Research;
Nicholas Smith, Powered Reserach; Brian C. Gilger, Powered
Research (C)
These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/
to access the versions of record.
ARVO 2016 Annual Meeting Abstracts
Program Number: 1287 Poster Board Number: D0235
Presentation Time: 3:15 PM–5:00 PM
A novel ex vivo model of equine corneal wound healing
Rita Wehrman, Joseph Haynes, Gil Ben-Shlomo. Veterinary Clinical
Sciences, Iowa State University, Ames, IA.
Purpose: To develop an ex vivo model of equine corneal epithelial
wound healing while maintaining normal corneal anatomy.
Methods: Equine corneas were harvested within two hours of
humane euthanasia (for reasons unrelated to this study) and
immediately processed. Corneoscleral rims were excised 2mm
posterior to the limbus. The axial cornea was wounded with filter
paper soaked in 1N NaOH and epithelial ulceration confirmed
employing fluorescein stain. Corneas were subsequently cultured
using an air-liquid interface model in media supplemented with 10%
fetal bovine serum, 10ng/ml human growth factor and 5ug/ml bovine
pancreatic insulin. The rocker was set to bathe the cornea 8 times
per minute to simulate normal horse blinking. Corneas were stained
with fluorescein and imaged every other day to assess wound healing.
Ulcer size was analyzed using an image processing program and
corneas were submitted for histologic evaluation when fluorescein
negative.
Results: All corneas healed within 4 days (96 hours) of epithelial
ulceration. Histologically, corneas maintained normal architecture
including viable epithelium and minimal stromal edema.
Conclusions: Air-liquid interface with media bathing is an effective
ex vivo model of equine corneal wound healing.
Commercial Relationships: Rita Wehrman, None; Joseph Haynes;
Gil Ben-Shlomo, None
Program Number: 1288 Poster Board Number: D0236
Presentation Time: 3:15 PM–5:00 PM
Neuropathic changes in corneal nerve fibers after abrasion injury
Sue Aicher, Clayton Hudson, Sam Hermes, Deborah Hegarty.
Physiology & Pharmacology, Oregon Health & Science University,
Portland, OR.
Purpose: Alterations in ocular sensation occur in patients with
dry eye disease as well as those who have undergone vision
correction surgery. Pain experienced by these patients may indicate a
neuropathic pain condition. We tested the hypothesis that neuropathic
changes in corneal nerve fibers underlie changes in ocular sensation
using a corneal abrasion injury in rats. These basic science studies
will determine the relationship between changes in corneal fiber
density and neurochemical content and ocular sensation after a
corneal injury.
Methods: A unilateral central corneal injury was made using
topical application of heptanol in male Sprague-Dawley rats. At
time points after corneal abrasion injury, stereotypic nociceptive
eye wipe responses to ocular application of noxious menthol were
measured in either the abraded eye or the non-abraded control eye.
At the conclusion of behavioral testing, rats were euthanized and
perfused with aldehydes. The corneas were removed and processed
for immunohistochemistry for beta-tubulin and nociceptive molecular
markers. Corneal fibers were visualized with confocal microscopy
and analyzed using Imaris software. T-tests were used for statistical
analyses.
Results: Rats tested in the injured eye 24 hours after abrasion had a
significantly higher average number of eye wipe responses to ocular
application of noxious menthol (13.8 ± 0.8 (SEM) eye wipes; n=4)
as compared to rats tested on the uninjured side (7.7 ± 0.9 (SEM) eye
wipes; n=3; t-test, p=0.002). Paradoxically, abraded corneas from the
24 hour time point had dramatically reduced corneal fiber density.
One week post-injury, rats tested on the injured side (9.3 ± 0.5 (SEM)
eye wipes; n=4) did not show any significant differences from rats
tested on the uninjured side (9.0 ± 2.3 (SEM) eye wipes, n=4). While
corneal fiber density showed evidence of recovery at one week, it was
reduced compared to uninjured corneas.
Conclusions: Abrasion injury reduces corneal fiber density; however
this reduction does not appear to be directly correlated to behavioral
responses to noxious corneal stimulation. We believe that the
neurochemical content of corneal fibers will be a better predictor
of pain sensitivity at the corneal surface than overall density. These
findings will provide insights into the dynamics of pain transmission
in the cornea and will deepen our understanding of the neuropathic
changes that occur in corneal nerve fibers after injury.
Commercial Relationships: Sue Aicher; Clayton Hudson, None;
Sam Hermes, None; Deborah Hegarty, None
Support: OHSU Presidential Bridge Funding
Program Number: 1289 Poster Board Number: D0237
Presentation Time: 3:15 PM–5:00 PM
Modulation of corneal wound healing in the mouse using
pre-surgical fasting
Matthew Petroll, Pouriska Kivanany. Ophthalmology, Univ Texas
Southwestern Med Ctr, Dallas, TX.
Purpose: Long-term dietary restriction (DR), defined as reduced
food intake without malnutrition, has been shown to extend lifespan
in rodents, as well as provide increased resistance to many forms of
acute stress. Long-term DR has also been shown to alter proliferation
and fibrosis during dermal wound healing. Increased longevity has
been shown to require long-term DR. However, recent studies have
established that short-term, water only fasting is sufficient to promote
many stress resistance responses. In this study, we investigate the
effect of short-term fasting on corneal wound healing in the mouse.
Methods: A transcorneal freeze injury (FI) model was used on
ten C57/BL6 mice. Animals were either fed ad libitum (5 mice) or
underwent 2 days of water only fasting (5 mice) prior to receiving a
1 mm diameter FI in the center of the left eye. After FI, all mice were
fed ad libitum. Wound healing was assessed from 3 to 28 days after
injury using 3-D in vivo confocal microscopy with a custom modified
HRT-Rostock Corneal Module. At 7 days, a subset of corneas was
fixed in situ, labeled for f-actin and nuclei, and imaged using confocal
fluorescence microscopy.
Results: The epithelium was resurfaced at 3 days after FI in both
fasted and non-fasted animals. In animals fed ad libitum, activated
stromal cells were observed in the anterior stroma 3 days after FI.
By 7 days, activated cells were present throughout the full thickness
of the corneal stroma. Short-term fasting inhibited the initial wound
healing response, as indicated by a lack of activated stromal cells 3
days after injury. At 7 days, stromal cell activation appeared similar
between fasted and non-fasted animals. F-actin labeling confirmed
that stromal cells expressed intracellular stress fibers at 7 days after
FI in both fasted and non-fasted animals. Stromal cell activation was
reduced at 14 and 21 days after injury in both fasted and non-fasted
animals.
Conclusions: These initial results suggest that short term fasting
reduces and/or delays the initial corneal stromal wound healing
response in the mouse. We hypothesize that the transient nature of
this response is due to the fact that the mice were refed at the time of
injury. We hypothesize that by using DR both before and after injury,
a sustained impact on wound healing could be achieved.
Commercial Relationships: Matthew Petroll, None;
Pouriska Kivanany, None
Support: NIH Grants R21 EY025057 and P30 EY020799, and
Research to Prevent Blindness, Inc.
These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/
to access the versions of record.
ARVO 2016 Annual Meeting Abstracts
Program Number: 1290 Poster Board Number: D0238
Presentation Time: 3:15 PM–5:00 PM
Inhibitor of Differentiation Gene: Expression, Localization and
Role in Human Cornea
Rajiv R. Mohan1, 2, Brandie R Morgan1, 3, Govindaraj Anumanthan1, 3,
Ajay Sharma1, 3, Shyam Chaurasia3, Prashant R. Sinha1, 3,
Frank G. Rieger1. 1Harry S. Truman Veterans Hospital, Columbia,
MO; 2Veterinary Medicine & Surgery, Biomedical Sciences,
Veterinary Pathology, and Mason Eye Institute, University of
Missouri, Columbia, MO; 3Veterinary Medicine & Surgery,
University of Missouri, Columbia, MO.
Purpose: Inhibitor of differentiation (Id) genes are DNA-binding
transcription factors and shown to regulate cell proliferation,
migration, angiogenesis and fibrosis. The expression and role Id
genes in the cornea are still unknown. We sought to characterize the
expression of Id proteins and their interactions with the profibrotic
cytokine transforming growth factor β1 (TGFβ1) and anti-fibrotic
cytokine bone morphogenic protein 7 (BMP7) in human cornea.
Methods: Human donor corneas procured from Eye Bank were
used. Id proteins were localized in human corneal sections using
immunofluorescence. Primary cultures of human corneal fibroblasts
(HCF) were established and treated with either TGFβ1 (5ng/ml) or
BMP7 (10ng/ml) for 24 hrs in serum free medium. Expression of Id’s
in response to TGFβ1, BMP7 and TGFβ1+BMP7 was measured with
qPCR and immunoblotting.
Results: All three major cell types of human cornea expressed
Id1-4 genes. Id1 and Id2 proteins were ubiquitously expressed in
epithelial cells and stromal keratocytes in the human cornea with Id1
predominantly in basal epithelial cells. The Ids were differentially
regulated with TGFβ1 and BMP7 in a time dependent manner. Id
proteins are exclusively expressed in human corneal fibroblasts but
not in human corneal myofibroblasts. TGFβ1 and BMP7 regulated
Id gene expression at different time points. Treatment of TGFβ1 to
HCF showed a significant increase in Id1, Id2 and Id4 at earlier time
point (2h; Id1, p<0.001; Id2, p<0.01 and Id4, p<0.001) followed by a
decrease in mRNA expression at late time points (12h, 24h, and 48h).
Contrary to this, BMP7 treatment showed a gradual increase in the
expression of Id1 (24h p<0.005; 48h p<0.001), Id2 (24h p<0.01) and
Id3 (48h p<0.001) in a time-dependent manner. Combined treatment
of TGFβ1+BMP7 to HCFs showed a significant increase in Id1, Id3
and Id4 expression.
Conclusions: Id genes are expressed in human cornea and play vital
role in corneal wound healing and fibrosis development by regulating
TGFβ1 and BMP7 activities. Id genes can be a target for restoring
and maintaining corneal transparency.
Commercial Relationships: Rajiv R. Mohan; Brandie
R Morgan, None; Govindaraj Anumanthan, None; Ajay Sharma,
None; Shyam Chaurasia, None; Prashant R. Sinha, None;
Frank G. Rieger, None
Support: 2RO1EY17294 National Eye Institute, NIH, Bethesda and
1I01BX000357-05 Veteran Health Affairs, Washington DC
Program Number: 1291 Poster Board Number: D0239
Presentation Time: 3:15 PM–5:00 PM
The Therapeutic Utility of Decorin Eye Drops for the Prevention
of Corneal Scarring
Maryam Esmaeili1, Zubair Ahmed1, Saaeha Rauz2, Liam M. Grover3,
Ann Logan1. 1Neurotrauma, University of Birmingham, Birmingham,
United Kingdom; 2Opthalmology, University of Birmingham,
Birmingham, United Kingdom; 3Biochemical Engineering, University
of Birmingham, Birmingham, United Kingdom.
Purpose: Corneal opacity is a leading cause of worldwide blindness.
The availability of drugs to treat established corneal scars or the
prevention of scar formation following trauma, is limited. We have
previously shown that Decorin, a naturally occurring antagonist
of TGF-beta, creates a permissive microenvironment that enables
healing with reduction of pro-fibrotic cascades. In this study, we
examined the use of Decorin eye drops as an early stage intervention
to reduce visually significant corneal scar formation in a corneal
injury animal model.
Methods: Decorin was delivered as a single 40µL drop onto the
corneal surface of anaesthetised adult Sprague-Dawley rats and
penetration into the cornea and aqueous humour (AqH) was assessed
at 0, 30, 60 min by immunohistochemistry and ELISA, respectively.
Eyedrop deliverable levels of Decorin were compared with those
present endogenously in human AqH. An ex-vivo rat corneal-scarring
alkali burn model was established by exposing the central cornea to
filter paper saturated with 0.5M NaOH to varying time points (15,
30 and 60 s) followed by histological analysis of tissue for corneal
scarring. The topical application of Decorin was evaluated.
Results: Decorin penetrated the rat cornea into the anterior segment
of the eye. While Decorin levels in corneal tissue increased over
time, concentrations in the AqH decreased (10 min, 12600pg/
mL (±3382pg/mL (SEM); n=3) to 60 min, 4600pg/mL (±3494pg/
mL (SEM); n=3)). This level was comparable to physiological
concentrations of Decorin in human AqH (1904pg/mL (± 360pg/
mL (SEM); n=10). Topical Decorin is able to prevent corneal scar
formation in our ex vivo rat corneal-alkali injury model compared to
eyes that were not treated with Decorin.
Conclusions: Topical application of Decorin is effective in reducing
corneal scar formation during acute alkali injury and is likely to be
therapeutically useful in the prevention of corneal scarring. Anteriorsegment penetration of Decorin may afford a novel anti-fibrotic
strategy for intraocular scarring processes.
Commercial Relationships: Maryam Esmaeili, None;
Zubair Ahmed, None; Saaeha Rauz, None; Liam M. Grover,
None; Ann Logan, None
Support: Wellcome Trust
These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/
to access the versions of record.
ARVO 2016 Annual Meeting Abstracts
Program Number: 1292 Poster Board Number: D0240
Presentation Time: 3:15 PM–5:00 PM
Subbasal Axon Density is Reduced and Reinnervation After
Injury Delayed in the Syndecan-1 Null Mouse Cornea
Mary Ann Stepp, Gauri Tadvalkar, Raymond Hakh,
Sonali Pal-Ghosh. Anatomy and Regenerative Biology, George
Washington University, Washington, DC.
Purpose: The heparan sulfate proteoglycan syndecan is required for
proper growth cone formation in C. elegans; although syndecan-1
is upregulated in wounded sensory axons in the mouse, how loss of
syndecan-1 impacts subbasal sensory nerve (SBN) innervation and
reinnervation in the syndecan-1 null mouse cornea is not known.
Methods: The axon density of SBNs was quantified in unwounded
as well as 1.5 mm trephine-only and debridement wounded corneas
in wt and sdc1 null mice from 1-28 days after injury. For each wound
type and time point a minimum of 5 corneas were assessed. SBN
density was quantified in whole mount corneas stained to localize β3
tubulin. Confocal images were acquired and axon density quantified
using the Sholl analysis. Significance was assessed by ANOVA.
Results: SBN axon density in the center and periphery of unwounded
sdc1 null mice is reduced to 80% and 58% that of the control levels.
The SBNs in trephine-only (crush) wounded sdc1 null corneas
reinnervated slower over time than those in wt mice; in addition, after
small wounds, SBNs reinnervated significantly slower in sdc1 null
mice compared to wt mice after both wound types.
Conclusions: In unwounded sdc1 null mice, SBN density is
reduced compared to wt mice. However, the vortex is retained.
The SBNs of sdc1 null mice reinnervate slower after trephine
only and debridement wounds. Previous studies have shown that
reepithelialization of corneal debridement wounds of sdc1 null mice
is slower and that they develop fewer recurrent erosions. These
results show that sdc1 homologs (sdc2, sdc3, and sdc4) in the mouse
likely compensate for the loss of sdc1 in the development and
localization of SBNs in the mouse cornea.
Commercial Relationships: Mary Ann Stepp; Gauri Tadvalkar,
None; Raymond Hakh, None; Sonali Pal-Ghosh, None
Support: EY08512, EY021784, and EY023106
Program Number: 1293 Poster Board Number: D0241
Presentation Time: 3:15 PM–5:00 PM
Particulated extracellular matrix as immunomodulators for
corneal wound healing
Hongbo Yin1, 2, Qiaozhi Lu2, Xiaokun Wang2, Jennifer Elisseeff2.
1
Department of Opthalmology, West China hospital, Baltimore,
MD; 2Translational Tissue Engineering Center, Wilmer eye institute,
Baltimore, MD.
Purpose: Extracellular matrix (ECM) is essential during wound
repair, as it provides the structural integrity and regulates cellular
functions via the actions of cytokines and growth factors.
Corneal wound healing is a complex process that involves ECM
remodeling and immunomodulation. Here we reported the effects
of administering particulated decellularized ECM (pdECM) from
porcine lymph nodes in reducing corneal scarring after manual
superficial keratectomy (MSK).
Methods: Porcine lymph nodes were decellularized, lyophilized and
cryomilled to obtain microparticles. Young white rabbits were used as
the animal model and fibrin glue (FG) was administered on wounded
corneas as the standard treatment. For each rabbit, MSK was
performed on one eye to remove 150 μm of anterior cornea and the
other cornea was left unwounded as the healthy control. The animals
were divided into three groups: FG, FG encapsulated with pdECM
and no treatment. Photos of healthy and wounded corneal were taken
regularly to examine the epithelial healing and cornea haze. Corneal
samples were harvested at both one week and one month. Real-time
PCR and immunohistochemistry were performed to evaluate the
wound healing outcomes and scar formation.
Results: The reepithelialization was complete within 5 days in
all three groups. Treated groups had significantly reduced corneal
haze scores compared to untreated corneas, and the addition of
pdECM had even better effect than FG alone. pdECM was able to
even further decrease the expression of both inflammatory factors
(MMP9 and TNFα) at early stage (one week) and fibrosis-related
genes (collagen I, TGFβ1 and CTGF) at the later stage (one month)
compared to the FG group. pdECM could better maintain the
corneal thickness and the normal morphology of epithelium as
seen in H&E staining as well. Immunohistochemistry with CD11b
demonstrated fewer macrophages infiltrations with pdECM, which
also significantly downregulated the αSMA expression in regenerated
corneal stroma.
Conclusions: pdECM is promising in modulating corneal wound
healing after MSK via reducing the expression of proinflammatory
cytokines and regulating the migration of immune cells during the
wound healing process. pdECMplay a key role in modulating the
regenerative process and is potentially a new therapeutic in affiliating
the scarlesshealing after corneal surgery.
Commercial Relationships: Hongbo Yin; Qiaozhi Lu, None;
Xiaokun Wang, None; Jennifer Elisseeff, None
Program Number: 1294 Poster Board Number: D0242
Presentation Time: 3:15 PM–5:00 PM
Chemical burn induced stromal demarcation line
Koby Brosh. ophthalmology, Shaare zedek medical center, Jerusalem,
Israel.
Purpose: A stromal demarcation line is a well known sign after
collagen cross-linking (CXL)1-5. It has been proposed that this line
is the transition zone between cellular and acellular stroma and thus
might reveal the depth of photochemical changes in the corneal
stroma3-4. We report two cases of a similar demarcation line following
chemical alkali burns. To the best of our knowledge, this is the first
report of a stromal demarcation line following a chemical burn.
Methods: Two cases of chemical burn induced stromal demarcation
line are presented. Frequent AS-OCT follow-up was used in order to
demonstrate the demarcation line and its characteristics.
Results: Two patients arrived at the emergency room following an
ocular alkali burn. At presentation both had total corneal erosion,
corneal edema and limbal ischemia. Twelve to fifteen days later, a
stromal line was apparent by both slit lamp examination and anterior
segment OCT (AS-OCT). The stromal demarcation lines disappeared
approximately three months after the injury.
Conclusions: A stromal demarcation line may appear not only after a
CXL but also after a chemical burn. The line depth may be associated
with the severity of the injury, and therefore, may have prognostic
significance. Patients with chemical burns should be examined for
evidence of a stromal line in the corneal stroma.
Stromal demarcation line (white arrows) and CCT demonstrated by
horizontal OCT through the midline of the right cornea of patient 1.
These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/
to access the versions of record.
ARVO 2016 Annual Meeting Abstracts
Stromal demarcation line (white arrows) and CCT demonstrated by
horizontal OCT through the midline of the cornea (OD) of patient 2.
Commercial Relationships: koby Brosh, None
These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/
to access the versions of record.