jurnal descemetocele
TRANSCRIPT
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NEW STRATEGIES IN CANINE CORNEAL WOUND HEALING FOR THE VETERINARY
OPHTHALMIC PATIENT
_______________________________________
A Thesis
presented to
the Faculty of the Graduate School
at the University of Missouri-Columbia
_______________________________________________________
In Partial Fulfillment
of the Requirements for the Degree
Master of Science
_____________________________________________________
by
ANN P. BOSIACK
Dr. Elizabeth A. Giuliano, Thesis Supervisor
DECEMBER 2012
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The undersigned, appointed by the dean of the Graduate School, have examined the thesis
entitled
NEW STRATEGIES IN CANINE CORNEAL WOUND HEALING FOR THE VETERINARY
OPHTHALMIC PATIENT
presented by Ann Bosiack,
a candidate for the degree of master of science,
and hereby certify that, in their opinion, it is worthy of acceptance.
Professor Elizabeth A. Giuliano
Professor Jacqueline W. Pearce
Professor Cecil P. Moore
Professor Rajiv R. Mohan
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ACKNOWLEDGEMENTS
I want to express my deepest gratitude to Dr. Elizabeth Giuliano, my master’s
advisor as well as residency mentor. Her assistance and support have been instrumental to
the completion of this project. She contributed an enormous amount of her time to
helping me prepare and revise manuscripts and presentations. She is a patient and
supportive mentor who has provided me with exceptional guidance and instruction
throughout my residency and graduate career.
I would like to sincerely thank Dr. Rajiv Mohan for making this project possible.
He generously provided the laboratory, materials, and support needed to complete this
project. His collaboration with the ophthalmology service at the veterinary school has
been an invaluable aspect of my residency program. He helped extensively with the
experimental design and execution as well as with the manuscript revision and
submission. He has contributed an enormous amount of research in the corneal field of
medicine and it has been an honor to work with him.
I would also like to thank my other graduate committee members Dr. Jackie
Pearce and Dr. Cecil Moore. Their guidance and support throughout my residency made
it possible for me to accomplish a concurrent master’s degree. As residency mentors I
am, undeniably, ever grateful for all that they have taught me.
Lastly, I would like to acknowledge and thank Rangan Gupta and all of the other
members of Dr. Mohan’s laboratory for their support and assistance in the lab.
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TABLE OF CONTENTS
ACKNOWLEDGEMENTS ................................................................................................ ii
LIST OF ILLUSTRATIONS ............................................................................................. v
LIST OF TABLES ............................................................................................................. vi
Chapter
1. INTRODUCTION ....................................................................................................1
Canine Corneal Disease ......................................................................................1
Corneal Gene Therapy In Veterinary Medicine ...................................................2
Adeno-Associated Virus ......................................................................................4
Gene Transcription Modulators ...........................................................................5
2. GENE THERAPY IN COMPARATIVE OPHTHALMOLOGY ............................6
Vectors .................................................................................................................6
Potential Applications In Veterinary Medicine ....................................................9
Conclusions ........................................................................................................18
3. CANINE CORNEAL FIBROBLAST AND MYOFIBROBLAST
TRANSDUCTION WITH AAV5 ...........................................................................20Experimental Purpose ........................................................................................20
Materials And Methods ......................................................................................20
Results ................................................................................................................26
Discussion ..........................................................................................................28
4. EFFICACY AND SAFETY OF SUBEROYLANILIDE HYDROXAMIC
ACID (VORINOSTAT) IN THE TREATMENT OF CANINE CORNEALFIBROSIS ................................................................................................................32
Experimental Purpose ........................................................................................32
Materials And Methods ......................................................................................32
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Results ................................................................................................................37
Discussion ..........................................................................................................39
APPENDIX ........................................................................................................................43
1. FIGURES ................................................................................................................43
BIBLIOGRAPHY ..............................................................................................................58
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LIST OF ILLUSTRATIONS
Figure Page
1. Representative immunocytochemistry images demonstrating phalloidin
staining for F-actin (shown in red) in canine corneal fibroblasts (a) andmyofibroblasts (b). ......................................................................................................44
2. Representative images demonstrating delivered GFP staining in CCFs. .......................45
3. Representative images demonstrating delivered GFP staining in CCMs ........................46
4. Quantification of GFP positive cells showing the amount of gene delivery
by AAV5 into CCFs as well as CCMs. .....................................................................47
5. Quantitative PCR measurements of AAV5-delivered GFP in CCFs and
CCMs. ........................................................................................................................48
6. Phase-contrast microscopic images of CCF (a) and CCM (b) culturestaken 48 h after AAV transduction. ...........................................................................49
7. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)assay demonstrating apoptotic cells (pink staining) in CCF cultures
transduced with AAV5-GFP. Trypan Blue exclusion data (c) shows the
percent viability of CCFs 24 h and 48 h following AAV transductioncompared to controls. .................................................................................................50
8. A bar graph showing the cellular viability as determined by trypan blue
assay in order to demonstrate the dose dependent effect of SAHA onCCFs. ..........................................................................................................................51
9. A composite image showing phase-contrast microscopy images of CCFs. ....................52
10. Representative images of TUNEL assay results detecting apoptosis of
CCFs in the untreated control group (a) and in CCFs treated with
0.06% SAHA for 24 h (b). .........................................................................................53
11. Immunocytochemical staining using αSMA antibody. ..................................................54
12.
Bar graphs showing αSMA immunostaining quantification. ..........................................55
13. Bar graphs showing quantitative real-time PCR results demonstrating the
relative expression of αSMA. ....................................................................................56
14. Western blot analysis of αSMA protein. ........................................................................57
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LIST OF TABLES
Table Page
1. Characteristics of viral vectors used in corneal gene therapy…………………..43
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CHAPTER 1:
INTRODUCTION
1. CANINE CORNEAL DISEASE
Canine corneal fibrosis is a significant clinical problem in veterinary ophthalmology. The
cornea provides two thirds of the refractive power of the eye. Corneal transparency and clarity
rely on the special organization of the cornea's constituent components.1 Corneal injury and/or
infection often results in fibrosis2, which may cause significant visual impairment.
3
Corneal wound healing following injury or infection often results in decreased corneal
tissue transparency due to corneal fibrosis. The transformation of quiescent keratocytes to
corneal fibroblasts and myofibroblasts has been identified as the primary event in corneal wound
healing that is responsible for fibrosis. While the production of myofibroblasts is a normal part
of the complex process of corneal wound healing, the overproduction of myofibroblasts can lead
to vision loss due to corneal scarring.4
Canine ulcerative keratitis is a significant clinical problem in veterinary ophthalmology.3
Corneal fibrosis is a common outcome following complicated corneal ulceration and wound
healing. Treatment of deep corneal ulcers typically demands both medical and surgical
therapeutic strategies.5 Surgical procedures utilized for the repair of deep corneal ulceration
include conjunctival grafts, corneal grafts, corneo-conjunctival transposition, natural material
transplants, and application of tissue adhesives.6-9
These procedures and their associated
inflammatory response typically results in moderate to severe corneal scarring. Currently, no
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pharmacologic agents specifically aimed at reducing corneal fibrosis are routinely used in
veterinary ophthalmology.
2. GENE THERAPY IN COMPARATIVE OPHTHALMOLOGY
Almost all advances in ocular and vision research require the testing of new therapeutic
strategies in animal models prior to pilot use in clinical trials. In vitro studies are commonly
used prior to animal models in an effort to establish proof of principle and in some cases initial
drug doses. Animal models are then needed to determine efficacy and establish toxicity levels
associated with novel therapies. Although animal research is essential for furthering our
understanding of vision and the treatment and prevention of ophthalmic disease in people,
scientific advancement brought about through the use of animal testing is often slow to be
adopted for use in the veterinary clinical patient compared to its use in physician ophthalmology.
Leber Congenital Amaurosis (LCA) is an inherited degenerative retinal disease that
causes blindness in children. A majority of LCA cases are caused by mutations in the RPE65
gene. A naturally occurring RPE65 mutation in Briard dogs was found, similar to LCA in
people.10
A gene-knockout mouse model was produced, which in turn, greatly facilitated the
development of gene-replacement strategies. Pioneer efforts in gene therapy for congenital LCA
has demonstrated significant improvement of visual impairment in affected Briard dogs and is
being used to help restore vision in children.11
This well-known scientific advancement serves to
demonstrate the importance of animal models of ocular disease and the essential role they have
played in the discovery of underlying genetic defects, mechanism of disease, and development of
therapeutic strategies. While rodents are commonly used, use of larger animal models (i.e. dog,
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cat, pig) has become increasingly attractive to assess efficacy and safety of a variety of treatment
modalities that are being considered for clinical trials in people. The successful establishment of
a canine animal model for retinal diseases such as LCA is likely one important reason why
retinal gene therapy has advanced faster than corneal gene therapy.
Comparative ophthalmic disease shared among many different species has long been
recognized; however, recent international programmatical developments such as the One Health
Initiative have fostered improved collaborations among health science professions, especially
physicians and veterinarians. Similarities between the genomics and pathogenesis of many
ocular diseases shared between people and animals has encouraged a convergence of knowledge
between physician and veterinary ophthalmologists with significant potential to benefit the
prevention, treatment, and diagnosis of many ophthalmic diseases. The cornea represents one of
the most evolutionary conserved anatomic components across mammals with many shared
pathological processes in various species. Corneal gene therapy is therefore an ideal target for
collaborative biomedical research.
The potential therapeutic applications of gene therapy are vast. A major advantage of
gene therapy over the use of conventional drugs is the prospect of curing disease rather than
providing transient relief by suppression of disease symptoms. Replacing defective genes with
functional genes through the use of gene therapy offers the prospect for long-term therapeutic
benefits without repeat drug application. Successful vision restoration through the use of gene
therapy in patients affected with LCA has demonstrated the ability of gene therapy to potentially
cure ocular disease and prevent long-term blindness.12, 13
Further research in gene therapy is
needed to develop treatments for other vision-impairing ocular diseases.
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The cornea is an ideal target for gene therapy due to its accessibility, immune-privileged
nature, and ability to perform frequent non-invasive assessment.1, 14
Gene therapy has been
studied in animal models of ocular corneal graft rejection, neovascularization, wound healing,
fibrosis, macular degeneration, optic neuropathy, and retinal degeneration.1, 15-20
The success of
corneal gene therapy depends on the type of vector, the extent of therapeutic gene expression,
adverse immune responses, and the stage of intervention.21
3. Adeno-Associated Virus
Several viral as well as non-viral vectors have been developed to transport genetic
material into cells, each of which has advantages and limitations.1 Our previous studies found
Adeno-Associated Virus (AAV) to be a safe and efficient viral vector for transducing corneal
cells and delivering therapeutic genes in the cornea of a variety of species including rabbit,
mouse, human and horse.1, 14, 22-25
To the best of our knowledge, no study has been conducted
thus far to test the potential use of AAV for canine corneal gene therapy. Serotypes 1–9 of
AAV are most commonly used for gene therapy.16 The AAV vector possesses several important
properties that make it a highly successful vector for corneal gene therapy, including its ability to
transduce dividing and non-dividing cells, low pathogenicity, low immunogenicity, and long-
term transgene expression.1 Various studies have shown unique tropism and transduction
efficacy of different AAV serotypes for specific tissues.16
The efficacy of AAV2 and AAV5 to
deliver genes into the corneal stroma of rabbits and rodent in vivo, as well as the ability of
AAV6, AAV8, and AAV9 to deliver genes in mouse cornea in vivo and donor human cornea ex
vivo has been established.1, 16
The finding of variable transduction efficiency and tissue tropism
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has led to comparative studies quantifying transduction efficiency of various AAV serotypes in
corneal tissue. In a previous study, AAV5 was shown to have enhanced gene delivery when
compared to AAV2 in the rabbit cornea.14
4. GENE TRANSCRIPTION MODULATORS
Of the various cytokines that contribute to the development of corneal fibrosis following
injury, TGFβ1 has been found to play a critical role by facilitating the transdifferentiation of
corneal keratocytes to fibroblasts and myofibroblasts.2 It has been shown that inhibition of
corneal myofibroblast formation reduces corneal fibrosis and increases corneal transparency.26-29
Much research has been focused on pharmacologic agents that modulate TGFβ function and the
subsequent development of corneal fibrosis.
Recently, our lab demonstrated that Trichostatin A (TSA) effectively reduces haze and
scarring in vivo in the rabbit cornea without causing significant ocular side effects.30, 31
TSA is a
reversible inhibitor of HDAC (HDACi). Histone deacetylases (HDAC) increase the binding
affinity between histones and DNA and produces condensed chromatin, which decreases the
transcriptional activity of genes.32
Therefore treatment of mammalian cells with inhibitors of
HDAC activity, such as trichostatin A results in increased expression of a variety of genes.33
HDACs have been shown to be involved in fibrogenesis in a variety of organs. However, TSA is
still under clinical trial and is not routinely used in the clinical setting to treat patients. A
derivative of TSA, Vorinostat or suberoylanilide hydroxamic acid (SAHA) is a broad spectrum
HDACi. It is approved by the US Food and Drug Administration (FDA) for clinical use as a
chemotherapeutic agent for the treatment of cutaneous T-cell lymphoma.34
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CHAPTER 2:
CORNEAL GENE THERAPY IN VETERINARY MEDICINE: A REVIEW
1. VECTORS:
Conventi onal vectors:
Several viral as well as non-viral vectors have been developed to transport genetic
material into cells, each of which has advantages and limitations (table 1).1 A detailed
discussion of all currently used viral and non-viral vectors in ocular gene therapy is beyond the
scope of this review. Rather, this section of the chapter will serve to review the most common
vectors utilized for corneal gene therapy.
Several studies have assessed the efficacy of Adenovirus (AV) vectors for corneal gene
therapy.1 AV vectors have been shown to effectively deliver genes to the rodent cornea;
however, they provide short-term transgene expression and stimulate moderate to severe
inflammation.35, 36
Retroviral vectors can successfully deliver genes into the cornea but are not
capable of transducing non-dividing cells.37
Like adenovirus, retroviral vectors have also been
shown to stimulate mild-to-severe inflammatory responses and have oncogenic potential due to
their integration near proto-oncogenes, making them undesirable.1, 38
Lentiviral vectors appear
to be safer than retroviral vectors due to their lower risk of tumor oncogenesis .38
The origin of
lentiviral vectors are from equine infectious anemia virus and the human immunodeficiency
virus (HIV), thus safety remains a major concern. Attempts to improve safety of lentiviral
vectors include creating vectors that lack the entire viral genome.39
Disabled lentivirus vectors
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provide long-term transgene expression and are able to transduce slow/non-dividing cells,
making them better options for the transduction of corneal keratocytes and endothelial cells.
Adeno-Associated Virus (AAV) possesses several important properties that make it a
highly successful vector for corneal gene therapy including its ability to transduce dividing and
non-dividing cells, low pathogenicity, low immunogenicity, and long-term transgene
expression.1 Our previous studies found AAV to be a safe and efficient viral vector for
transducing corneal cells and delivering therapeutic genes into the cornea of rabbits, mice,
horses, and people.1, 14, 22-25
Serotypes 1–9 of AAV are most commonly used for gene therapy.16
Various studies have shown unique tropism and transduction efficacy of different AAV
serotypes for specific tissues.16
The finding of variable transduction efficiency and tissue
tropism has led to comparative studies quantifying transduction efficiency of various AAV
serotypes in corneal tissue. The efficacy of AAV2 and AAV5 to deliver genes into the corneal
stroma of rabbits and rodents in vivo, as well as the ability of AAV6, AAV8, and AAV9 to
deliver genes in mouse cornea in vivo and donor human cornea ex vivo has been established.1, 16
In a previous study, AAV5 was shown to have enhanced gene delivery when compared to AAV2
in the rabbit cornea.14
Non-viral gene therapy is the introduction of therapeutic genes via plasmid DNA into
target cells without the use of a virus. In general these techniques demonstrate low toxicity,
immunogenicity, and pathogenicity, rendering these techniques safer than viral methods.
However, they demonstrate low transduction efficiencies, which make them less desirable.
These techniques include microinjection, electroporation, sonoporation, gene gun, controlled
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corneal dehydration, laser, and chemical delivery methods. The reader is referred to a thorough
review of these techniques in a recent publication by Mohan et al.39
Contemporary vectors:
Tyrosine-mutant AAV
The AAV viral capsid plays a fundamental role in cellular tropism (i.e. binding and
uptake) thus contributing to the efficiency of transgene expression. When the AAV vector is
incorporated into the target cell it is vulnerable to normal cellular degradation pathways. By
directly mutating the tyrosine residues on the viral capsid, the degradation mechanisms of the
target cell are avoided, which leads to increased transduction.40
When injected subretinally or
intravitreally these tyrosine-mutant AAV vectors demonstrate several fold higher transgene
delivery in target retinal cells than non-mutant AAV vectors.41
The dose of tyrosine-mutant
AAV vector required for transduction is also significantly lower than that of AAV which lessens
the immune response.41
Nanoparticles
Nanoparticles (NPs) offer an alternative to the use of viral vectors in gene therapy. NPs
are particles that are 1-100 nm in size. There are several different forms of nanoparticles and
they typically contain a segment of DNA or RNA that is compacted with a polycationic
polymer .42
Due to their small size, NPs can readily interact with molecules on the cell surface or
inside cells. Unlike their viral vector counterparts, NPs do not introduce exogenous genes into
target cells. They tend to be less immunogenic and cytotoxic than viral vectors.43
Finally, NPs
are able to incorporate numerous ligands such as DNA, antibodies, peptides, and probes and
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therefore present an array of therapeutic modalities. Pathways of cellular internalization of NPs
include phagocytosis, macropinocytosis, clathrin- or caveolae-mediated endocytosis, and other
clathrin- and caveolae-independent endocytic pathways.42
There has been successful vector gene transfer with the use of NPs in phase I/II clinical
trials designed to treat cystic fibrosis.44
Limited studies have investigated the use of NPs for
ocular gene delivery.42, 45
Mohan et al. recently evaluated the safety and toxicity of gene transfer
technique using a 2-kDa polyethylenimine conjugated to gold nanoparticles (PEI2-GNPs) in the
human cornea in vitro and in rabbit cornea in vivo.45
PEI2-GNPs exhibited significant transgene
delivery in human corneal fibroblasts and did not appear to alter cellular viability or phenotype.
Rabbit in vivo studies revealed substantial gold uptake in the rabbit cornea with a slow clearance
of GNPs over time. Our results to date suggest that PEI2-GNPs appear safe for use in the cornea
and may have potential use for corneal gene therapy; however, further research examining the
clearance of GNPs and their effects on the optical properties of the eye is needed.45
2. POTENTIAL APPLICATIONS IN VETERINARY MEDICINE
Corneal disease is a commonly encountered clinical problem in veterinary medicine. The
cornea provides two thirds of the refractive power of the mammalian eye. Therefore corneal
disease that results in disruption of corneal health (e.g. clarity or refractive properties) can lead to
significant visual impairment. Unique properties of the cornea that enable this living tissue to be
essentially optically clear render it susceptible to limited pathologic responses to injury and often
opacification results. Some of the common corneal diseases encountered in veterinary medicine
include keratitis (i.e. ulcerative, immune-mediated, keratoconjunctivitis sicca (KCS)), neoplasia,
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corneal dystrophy, dermoids, chemical burns, lacerations, and corneal degeneration, among
others. We now present an overview of the potential applications of gene therapy for treatment
of several common corneal diseases in veterinary medicine.
Corneal Transplantation
In physician ophthalmology, by virtue of well-maintained and highly regulated eye-
banks, viable full-thickness corneal transplants are commonly used. In veterinary medicine the
use of fresh corneal transplants is limited for a number of reasons. First, harvesting and
preservation requirements of donor corneal tissue is not feasible in the majority of veterinary
practices.46 Second, tissue typing and donor-recipient rejection concerns are not well
understood. Third, medical-legal implication/permission of harvesting fresh, healthy donor
cornea from a euthanized dog has not been established. Presently, veterinary ophthalmologists
use frozen corneal transplants to provide tectonic support rather than optical clarity, as corneal
endothelial cells do not survive the freezing process.46-48
Common ocular conditions that may be
treated with a previously frozen corneal transplant include descemetoceles, stromal abscesses,
inflammatory keratitis with or without malacia, corneal perforation/iris prolapse, and feline
corneal sequestra. 46, 48, 49
Corneal graft rejection involves the recognition of foreign antigens from the donor graft,
which leads to an immune response by the host. Graft rejection is manifested by a loss of graft
transparency after an initial time period of graft clarity (approximately 2 weeks in people and
several days in horses).47, 50
Corneal vascularization, inflammation, and/or infection in the host
species prior to transplantation significantly increases the chance of corneal graft rejection or
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failure.50 Human patients with these pre-existing corneal conditions are considered high-risk
patients. Most veterinary patients that receive corneal transplants would therefore be considered
high risk as almost all conditions in which corneal transplantation is indicated in veterinary
medicine are characterized by these pre-existing conditions. The most important risk factor for
graft rejection is host cornea vascularization.50
In stark contrast to physician ophthalmology,
corneal vascularization is usually considered a desired healing response since vascularization
substantially improves the patient’s ability to resist corneal infection. Thus, while the vast
majority of the corneal transplants performed in veterinary ophthalmology undergo “rejection”
by physician standards, vascularization of the donor cornea is considered an optimal progression
in wound healing in the majority of cases.46, 47
Prevention of corneal graft rejection currently is achieved by suppressing the host
immune response. In humans, this is done with liberal use of systemic and topical
corticosteroids as well as cyclosporine. In veterinary medicine the use of corticosteroids is
contraindicated in the face of corneal infection, which is present in many of the patients that
undergo corneal transplantation.51
Therefore the development of an alternative therapy to
corticosteroids, such as gene therapy, to prevent corneal graft rejection and the resultant long-
term corneal fibrosis would be advantageous in veterinary medicine.
The unique setting afforded by having an established tissue bank for corneal transplants
is ideal for gene therapy, as donor cornea can easily be manipulated, ex vivo, prior to
transplantation. Several gene therapy methods have been examined in an effort to improve graft
survival by the delivery of therapeutic genes that modulate cellular apoptosis, angiogenesis, and
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wound healing.52
One study investigated the delivery of an intracellular enzyme, indoleamine
2,3- dioxygenase (IDO), using a lentivirus vector in murine corneal endothelium.53
Once
activated by cytokines, IDO leads to the immunoregulatory catabolism of tryptophan. The
reduction of tryptophan is thought to halt activated T cells in the cell cycle, which encourages
immune tolerance of the transplanted cornea. This same research group also evaluated the effect
of IDO transduction on donor corneas ex vivo prior to transplantation and found significant
prolongation of corneal allograft survival compared to controls.54
Another group found that the transfer of an anti-apoptotic gene, bcl-xL, using a lentivirus
vector was effective at inhibiting apoptosis in the corneal endothelium in vitro in a rodent model,
as well as significantly enhancing corneal graft survival in vivo.55
Another application of gene
therapy use for corneal transplantation involves the preservation of corneal endothelial cell
density in corneal tissue that is stored at eye banks. One study demonstrated that the over-
expression of transcription factor E2F2, using recombinant adenovirus ex vivo produced an
increased endothelial cell count in rabbit and human corneas.56, 57
The E2F2 over-expression
induced cell-cycle progression of corneal endothelial cells, which are normally amitotic, in the
first week after transduction and without inducing significant apoptosis. This therapy has great
potential for use in veterinary ophthalmology, since it is more practical to use corneas that have
been preserved long-term. This type of gene therapy may prove especially beneficial in the
treatment of corneal endotheliopathies.
Corneal F ibrosis/ Modulation of Corneal Wound Healing
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Corneal transparency and clarity rely on the special organization of the cornea's
constituent components including corneal collagens and the extracellular matrix.1 Corneal
wound healing following injury and/or infection often results in fibrosis/scarring2, which may
cause significant visual impairment.58
Much of the focus on the human side of corneal gene
therapy for treatment of corneal fibrosis is geared towards reduction of photorefractive
keratectomy-induced laser injury to the cornea. In veterinary ophthalmology corneal fibrosis
(scar) is most commonly an outcome following complicated corneal ulceration and wound
healing. As previously discussed, because vascularization is a desired component of the healing
response in many veterinary cases that would benefit from corneal transplantation, the role of
gene therapy might better serve to address the fibrosis that results from graft rejection.
Ulcerative keratitis is a significant clinical problem in veterinary ophthalmology.58
Treatment of deep corneal ulcers typically demands both medical and surgical therapeutic
strategies.5 Surgical procedures utilized for the repair of deep corneal ulceration include
conjunctival grafts, corneal grafts, corneo-conjunctival transposition, natural material transplants,
and application of tissue adhesives.6-9
These procedures and their associated inflammatory
response typically result in moderate to severe corneal scarring. Currently, pharmacologic
agents that are specifically aimed at reducing corneal fibrosis are not routinely used in veterinary
ophthalmology.
The transformation of quiescent keratocytes to corneal fibroblasts and myofibroblasts has
been identified as the primary event in corneal wound healing that is responsible for fibrosis.
While the production of myofibroblasts is a normal part of the complex process of corneal
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wound healing, the overproduction of myofibroblasts can lead to vision loss due to corneal
scarring.4
Our lab investigated the safety and efficacy of AAV5 for delivering gene therapy incanine corneal fibroblasts (CCFs) as a model of the pre-fibrotic state as well as in canine corneal
myofibroblasts (CCMs), as a model of corneal fibrosis. Our results demonstrated that AAV5 is
an effective vector for gene therapy in canine corneal fibroblasts and myofibroblasts in vitro.
The transduction rate in CCFs was 42.8% CCFs and 28.0% in CCMs. The tested AAV5 vector
showed significantly less transduction of CCMs compared to CCFs. This difference in
transduction efficiency of AAV5 between the two cell types may suggest that higher doses of
vector are required for delivering therapeutic genes into fibrotic tissue and it may signify the
importance of therapeutic strategies aimed at initial prevention of corneal fibrosis versus
treatment of established fibrosis. However, other potential causes of this noted transduction
efficiency difference may be due to the specific cell tropism of the viral vector. Future studies
are underway to investigate different AAV serotypes in order to determine if a particular
serotype demonstrates specific tropism for canine corneal myofibroblasts.
Of the various cytokines that contribute to the development of corneal fibrosis following
injury, TGFβ1 has been found to play a critical role by facilitating the transdifferentiation of
corneal keratocytes to fibroblasts and myofibroblasts.2 It has been shown that inhibition of
corneal myofibroblast formation reduces corneal fibrosis and increases corneal transparency.26-29
Therefore, it is thought that inhibiting TGFβ and/or its signal transduction may be a useful
strategy to regulate uncontrolled corneal healing and to prevent or treat corneal scarring. Much
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research has been focused on gene therapy approaches to modulate TGFβ function and the
subsequent development of corneal fibrosis.
Decorin, a small proteoglycan, which is a natural inhibitor of TGFβ has been successfully
delivered to the cornea by the use of gene therapy. Mohan et al. recently showed that decorin
gene transfection inhibits TGFβ-driven elevated expression of profibrogenic genes and
transformation of corneal fibroblasts to myofibroblasts in human corneal fibroblast cultures.59
Our laboratory also demonstrated a significant inhibition of corneal scarring in vivo in the rabbit
with the use of AAV5-mediated decorin gene therapy and did not detect any adverse effects at a
time point of one-month following transduction.
60
This was the first study to establish the
therapeutic potential of decorin gene therapy for treating corneal diseases. Currently, long-term
studies are underway to assess whether the over-expression of decorin will cause detectable
ocular complications or changes in corneal function or transparency.39
The ability of gene therapy to prevent corneal scarring in experimental animals has been
demonstrated in several previous studies as well. Seitz et al. evaluated the use of retroviral
vector-mediated genetic transduction of rabbit keratocytes in vivo modulating keratocyte
proliferation and decreasing corneal scarring.61
They employed the use of a suicide gene and
pro-drug combination of the herpes simplex virus thymidine kinase (HSVtk) gene with
ganciclovir. HSVtk triggers cell death in proliferating keratocytes by converting ganciclovir to a
toxic metabolite. This combination gene therapy reduced corneal scarring 10 and 21 days post
administration. Galiacy et al. utilized an AAV vector expressing a fibril collagenase, matrix
metalloproteinase 14 (MMP14), in a murine model of corneal scarring.62
They observed that
MMP14 gene delivery reduced corneal opacity and expression of several major genes involved
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in corneal scarring. These findings are encouraging with regards to new developments for the
prevention and treatment of corneal fibrosis with possible direct application to the veterinary
ophthalmic patient.
Corneal Dystrophies
Corneal stromal dystrophy refers to a group of inherited corneal diseases that are
typically bilateral, symmetric and slowly progressive, with absent inflammation.63
They are
caused by an accumulation of material such as lipid or cholesterol crystals in the cornea. There
are a several forms of corneal dystrophy, which vary in their expression, location within the
cornea, and degree of vision loss. The dog is the most common nonhuman species that is
affected by corneal dystrophy and the condition is rare in other species. This condition manifests
as bilateral crystalline or white opacities in the central or paracentral cornea.64
Specific breeds
that are described in the literature to be affected by stromal dystrophies include the Shetland
Sheepdog, Beagle, Siberian Husky, Cavalier King Charles Spaniel, Airedale Terrier and Rough
Collie.65
In people the resulting corneal opacity causes visual impairment. In dogs, stromal
dystrophies infrequently lead to significantly appreciable functional vision loss. Nevertheless,
complete vision loss secondary to corneal dystrophy has been reported in Airedale Terriers and
Siberian Huskies.63
Boston Terriers, Chihuahuas and Dachshunds are affected by corneal
endothelial dystrophy, and the American Cocker Spaniel has been reported to develop a posterior
polymorphous dystrophy.65
Endothelial dystrophies (except the posterior polymorphous
dystrophy in the American Cocker Spaniel) are characterized by progressive corneal edema,
which can lead to vision loss, bullous keratopathy, and corneal ulcers.63
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Several genetic mutations in people with corneal dystrophies have been identified.66, 67
The pathogenesis and localization of many additional genes likely responsible for these corneal
diseases have yet to be identified. A paucity of research in the realm of gene therapy for the
treatment of corneal dystrophies exists for several reasons. There is a lack of animal models for
most of the corneal dystrophies described in people. The few mouse lines that have been created
to express corneal dystrophy genetic mutations do not exhibit similar clinical manifestations of
these diseases as observed in people.68
Additionally, the common use of corneal transplantation
as a relatively effective treatment in people may imply that corneal dystrophies are less visually
debilitating than other ocular diseases for which no treatment exists and as such, limited research
funding may be available to study corneal dystrophies at this time. Specific gene localization
and targeting could be vital for the development of animal models of this category of corneal
disease and for gene therapy treatment advances.
As is the case in physician ophthalmology, in veterinary ophthalmology there is a lack of
research focused on treatment of corneal dystrophies. Corneal dystrophies generally do not
respond to medical treatment; therefore, unless they cause an obvious loss of vision or have
complications (e.g. corneal ulceration) they are often treated with benign neglect. Treatment of
progressive corneal edema with endothelial dystrophy is palliative. When dogs develop
recurrent ulcers as a complication of dystrophies or persistent bullous keratopathy secondary to
endothelial dystrophy they may benefit from thermokeratopasty or keratoleptynsis.65, 69
Definitive treatment includes a living (i.e. one with functional donor corneal endothelial cells)
corneal transplant as previously described but is associated with relatively high failure rates in
dogs. Presently, there are no treatment strategies utilizing gene therapy for corneal dystrophies
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in veterinary ophthalmology. The corneal tissue-selective gene therapy approaches that are
currently under investigation may have potential for the treatment of corneal dystrophies in dogs.
Other
Mucopolysaccharidosis (MPS) is a group of lysosomal storage diseases characterized by
the defective metabolism of glycosaminoglycans (GAG). Five types of MPS have been
described in dogs (MPS I, II, III, VI, VII). While MPS is relatively rare in veterinary medicine,
the use of dogs in research is not uncommon because they serve as a large animal model for
human disease. Corneal pathology typically involves opacification of the cornea due to
lysosomal storage in keratocytes.52 Intravenous gene therapy in neonatal MPS dogs using a
retroviral vector has been shown to prevent GAG accrual and resultant corneal clouding.70, 71
Corneal clouding in untreated MPS I dogs resulted in a blurry view of the fundus as well as a
granular appearance of the cornea due to the lysosomal storage. A marked reduction in corneal
clouding was shown with systemic gene therapy using a retroviral vector in neonatal MPS I
dogs.70
3. CONCLUSIONS:
Animal models represent the corner stone of gene-therapy research. Many of the
advancements made to date have been possible through the appropriate use of rodents, rabbits,
dogs, cats, pigs, and non-human primates. Given the obvious comparative ophthalmic
implications to many of the recent scientific discoveries in vision science, the potential for
translational research to directly impact our veterinary ophthalmic patients is exciting. Our
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laboratory’s recent research in canine and equine corneas in vitro as well as significant progress
and advancement in the field of corneal gene therapy in the last decade holds promise for
establishing important models and for the improvement of long-term vision in our veterinary
patients and thus, improvement of human and animal welfare.
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CHAPTER 3:
CANINE CORNEAL FIBROBLAST AND MYOFIBROBLAST
TRANSDUCTION WITH AAV5
1. EXPERIMENTAL PURPOSE
The purposes of this study were to determine the efficacy of AAV5 for delivering
therapeutic genes in both normal and fibrotic canine keratocytes and to study the safety of AAV5
for the canine corneal gene therapy using an in vitro model of canine corneal fibroblasts (CCFs)
and myofibroblasts (CCMs).
2. MATERIALS AND METHODS
Canine corneal fibroblast and myofibroblast cultu res
All experimental procedures were approved by the Institutional Animal Care and Use
Committee of the University of Missouri. Ten full-thickness 6 mm axial corneal buttons were
aseptically harvested immediately post-mortem from five healthy research dogs undergoing
humane euthanasia for reasons unrelated to this study. Slit-lamp biomicroscopy was performed
by an experienced veterinary ophthalmologist (EAG) prior to euthanasia to ensure that all
samples were harvested from dogs free of anterior segment disease. The corneal biopsy samples
were washed with minimal essential medium (MEM, Gibco; Grand Island, NY, USA) and the
epithelium and endothelium were removed by careful manual scraping using a # 10 Bard Parker
scalpel blade (BD, Franklin lakes, NJ, USA). The corneal stroma was sectioned into four
smaller explants and placed in 100 X 20 mm tissue culture plates (BD BioSciences, Durham,
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NC, USA) containing MEM supplemented with 10% fetal bovine serum (Gibco, NY, USA), then
incubated in a humidified CO2 incubator (Thermo Scientific, Asheville, NC, USA) at 37oC in
order to obtain CCF cultures. In approximately 3-10 days primary fibroblasts began migrating
from the stromal sub-sections. On attaining ~90% confluence, the explants were manually
removed using rat-toothed forceps, and discarded. The confluent cells were trypsinized and the
cellular concentrations were calculated using Countess automated cell counter (Invitrogen) and
replated in 100 mm tissue culture dishes. Passages 1-3 of CCFs were used for this experiment
and all experiments were repeated in triplicate. Serum-free MEM medium containing 1ng/mL
TGFβ1 (PeproTech Inc, Rocky Hill, NJ, USA) was used to generate CCM cultures from
fibroblasts.
AAV vector production
Production of AAV5 expressing Enhanced Green Fluorescent Protein (GFP) under
control of hybrid cytomegalovirus (CMV) + chicken (β-actin (CBA) promoters (AAV5-gfp) was
performed by the gene therapy core, University of Florida, Gainesville, Florida and was procured
from Drs. Gregory S. Schultz and Vince A. Chodo. The AAV2 plasmid pTRUF11 expressing
green fluorescent protein gene under control of a hybrid promoter (cytomegalovirus enhancer
and chicken β-actin) and simian virus 40 polyadenylation site was packaged into AAV5. In
brief, AAV titer expressing GFP was produced by the 2-plasmid, co-transfection method 29.
One Cell Stack (Corning Inc., Corning, NY, USA) with approximately 1 X 109 HEK 293 cells
was cultured in Dulbecco’s Modified Eagle’s Medium (Hyclone Laboratories, Inc. Logan UT,
USA), supplemented with 5% fetal bovine serum and antibiotics. A calcium phosphate
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transfection precipitation was set up by mixing a 1:1 molar ratio of AAV2 plasmid DNA and
AAV5 rep–cap helper plasmid DNA. This precipitate was applied to the cell monolayer and the
transfection was incubated at 37°C, at 7% CO2 for 60 h. The cells were then harvested and lysed
by freeze/thaw cycles and subjected to discontinuous iodixanol gradient-ultra centrifugation at
350,000g for 1 hour (h). This iodixanol fraction was further purified and concentrated by
column chromatography on a 5-ml HiTrap Q Sepharose column using a Pharmacia AKTA FPLC
system (Amersham Biosciences, Piscataway, NJ, USA). The vector was eluted from the column
using 215 mM NaCl buffer, pH 8.0, and the rAAV peak collected. The AAV5-GFP vector-
containing fraction was then concentrated and buffer exchanged in Alcon BSS with 0.014%
Tween 20, using a Biomax 100K concentrator (Millipore, Billerica, MA, USA). The vector was
titered for DNAse-resistant vector genomes by Real-Time PCR relative to a standard.
AAV5 transduction
Seventy-percent confluent CCF or CCM cultures in 12-well plates were used for all
experiments. Cultures were serum deprived for 2 h prior to viral transduction (viral titer of
6.5x1012
genomic copies/mL). AAV5 expressing GFP marker gene (2 μL for each well of a 12
well plates) was applied in serum-free MEM medium. After 6 h, the medium containing viral
vector was replaced with fresh medium supplemented with 10% serum and the cells were
incubated for 48 h prior to microscopic evaluation of fluorescence.
Cytotoxi city Studies
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The toxicity and safety of AAV5 for canine corneal gene therapy was evaluated by
comparing phenotype, cell death, and cellular viability in AAV5-treated and untreated CCFs and
CCMs. Phase-contrast light microscopy images were obtained 24 h and 48 h after application of
AAV5 to observe any phenotypic changes in CCF and CCM cultures. Terminal
deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay was used
to detect cellular apoptosis due to AAV vector transduction in CCFs and CCMs. Cells were
fixed with 4% paraformaldehyde at room temperature for 15-20 min and then washed with
phosphate buffered saline. A fluorescence-based TUNEL assay was used according to the
manufacturer’s instructions using ApopTag apoptosis detection kit (Chemicon international,
Temecula, CA, USA). Cellular viability was assessed using trypan blue (Sigma-Aldrich, St.
Louis, MO, USA) exclusion 24 h and 48 h after transduction. Cell count was performed using an
automated cell counter.
Immunocytochemistry
Immunocytochemical analysis was performed in order to determine transduction
efficiency. Direct fluorescence microscopy at 100X magnification was used to count GFP
transduced CCFs and DAPI-stained nuclei in ten randomly selected, non-overlapping areas for
each treatment. Analysis was performed on a PC computer using the public domain NIH Image
JAVA program (developed at the U.S. National Institutes of Health and available on the Internet
at http://rsb.info.nih.gov/nih-image). Percent transduction efficiency was calculated by dividing
the total number GFP-positive cells by the total number of 4',6-diamidino-2-phenylindole
(DAPI) stained nuclei.
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Immunofluorescent staining for αSMA, a marker for myofibroblasts, was performed
using mouse monoclonal antibody for and αSMA. At study end point, cultures were washed
twice with PBS and incubated at room temperature with mouse monoclonal antibody for αSMA
(DAKO, Carpinteria, CA, USA) at a 1:200 dilution in 1X PBS for 90 min. Cultures were then
washed twice with PBS and the incubated at room temperature with secondary antibody Alexa
488 goat anti-mouse IgG (Invitrogen, Carlsbad, CA, USA) at a dilution of 1:500 for 1
h. Immunofluorescent staining for F-actin, which stains cytoskeletal cellular structures, was
performed using phalloidin (Invitrogen, Calsbad, CA, USA). At study end point, cultures were
washed twice with PBS and incubated at room temperature with phalloidin (Invitrogen, Calsbad,
CA, USA) at a 1:200 dilution in 1X PBS for 90 min. Cells were mounted with Vectashield
containing 4’-6’-Diamidino-2-phenylindole (DAPI) (Vector laboratories, Inc., Burlingame, CA,
USA) to allow visualization of nuclei. Irrelevant isotype-matched primary antibody, secondary
antibody alone, and tissue sections from naïve eyes were used as negative controls for each
immunocytochemistry experiment.
Immunostained cultures were examined and photographed with Leica fluorescent
microscope (Leica, Wetzlar, Germany) equipped with a CCD digital camera (SpotCam RT KE,
Diagnostic Instruments Inc., Sterling Heights, MI, USA). The immunocytochemistry data
quantification was performed by counting GFP, TUNEL, and DAPI-positive colored cells in 10
randomly selected non-overlapping areas of fluorescent cultures at 100X magnification using a
Leica Fluorescent microscope (Leica).
RNA extr action, cDNA synthesis, and quantitative real-time PCR
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The total RNA from CCF and CCM cultures transduced with AAV5-GFP was extracted
using RNeasy kit (Qiagen Inc., Valencia, CA, USA) and reverse-transcribed to cDNA following
manufacturer’s instructions (Promega, Madison, WI, USA). Real-time PCR was performed
using iQ5 real-time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA). A 20
µL reaction mixture containing 2 µL cDNA, 2 µL forward (200nM), 2 µL reverse primer
(200nM), and 10 µL 2X iQ SYBR green super mix (Bio-Rad Laboratories) was run at universal
cycle (95oC 3 min, 40 cycles of 95
oC 30 sec followed by 60
oC 60 sec) following manufacturer’s
instructions. For GFP, forward primer sequence GCGTGCAGTGCTTTTCCAGA and reverse
primer sequence CTTGACTTCAGCGCGGGTCT were used. The house-keeping gene beta
actin forward primer sequence was CGGCTACAGCTTCACCACCA and reverse primer
sequence was CGGGCAGCTCGTAGCTCTTC. The threshold cycle (CT) was used to detect the
increase in the signal associated with an exponential growth of PCR product during the log-linear
phase. The relative gene expression was calculated using the following formula: 2-ΔΔC
T. The CT
is a PCR cycle number at which the fluorescence meets the threshold in the amplification plot
and ΔCT is a subtraction product of target and housekeeping genes CT values. The 2-ΔΔCT is a
method for determining relative target mRNA quantity in samples. The amplification efficiency
for all used templates was validated by ensuring that the difference between linear slopes for all
templates <0.1. Three independent reactions were performed and the average (± standard error of
the mean or SEM) results were calculated.
Quant if ication and statistical analysis
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Results are expressed as mean ± standard error of the mean (SEM). Statistical analysis was
performed using analysis of variance (ANOVA) followed by Bonferroni test for comparing data
between control and AAV vector. Data was tested for normality using Kolmogorov-Smirnov
Test and probability plots. Microsoft excel and Graph Pad Prism statistical software was used.
3. RESULTS
AAV Transduction Ef fi ciency
Figure 1 shows establishment of canine corneal fibroblast (representing early healing
state) and myofibroblast (representing late healing state) monolayer cultures using phalloidin (F-
actin) immunostaining. The cells grown in 10% serum supplemented medium reveal classic
spindle shaped fibroblast morphology (Figure 1a); cells grown in serum-free medium containing
TGF1 (1ng/ml) show large and wide morphology typical of the myofibroblast phenotype
(Figure 1b). The growth kinetics of these cells was similar to previously reported findings of
equine corneal cells except canine corneal cells grew at a rate of about 2.5-3 fold faster .72
Successful transduction of GFP with AAV5 vector into CCFs and CCMs was determined
via fluorescent microscopy (Figures 2 and 3, respectively). The dual immunostaining
experiments were performed to confirm transgene delivery into the two cell types.
Myofibroblast marker, αSMA, is shown in red. Detection of ~ 3-5% myofibroblast cells in
corneal fibroblast cultures (Fig 2a) is normal and unavoidable.72
CCFs treated with AAV5-GFP
(Figure 2b) demonstrated significant transduction as evident from GFP-positive cells (green
fluorescence showing spindle shaped morphology) when compared to the untreated control cells
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(Figure 4a) indicating successful and efficient GFP gene transduction. Likewise, myofibroblasts
treated with AAV5-GFP (Figure 3b) demonstrated a significantly high of number of GFP-
positive cells compared to the untreated control cells (Figure 3a).
Quantification of GFP-positive CCFs and CCMs was completed at 100X magnification
following previously described methods (Figure 4). The AAV5-GFP vector transduced 42.8%
CCFs and 28.0% CCMs. The tested AAV5 vector showed significantly less (15%; p≤ 0.01)
CCM transduction compared to CCFs.
Quantif ication of real-time PCR
The relative delivery of the GFP gene in AAV5-treated CCFs and CCMs and their
respective controls was quantified using real-time PCR (Figure 5). In both cell populations there
is significant transduction of AAV5-GFP compared to the untreated controls. Percentage of
GFP-mRNA was significantly lower in CCM compared to CCF cultures.
AAV Safety
No observable changes in phenotype were detected in phase-contrast light microscopy
images between untreated control cells and AAV5-treated cells at 24 h and 48 h after the
application of AAV5. Figure 6 shows CCFs and CCMs 48 h after the application of AAV5.
These images demonstrate the classic morphology of CCFs and CCMs as well as the successful
progression to confluence. Similar results were seen at 24 h (data not shown).
TUNEL assay was used to detect CCF and CCM apoptosis, and to a lesser extent
necrosis. No detectable difference in cellular apoptosis was observed between AAV5-GFP
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treated CCFs and untreated controls (Figure 7a and 7b). Similar results were seen for CCMs
(results not shown).
Trypan blue exclusion assay demonstrated that cellular viability of the AAV5-GFP
treated CCFs was greater than 95% at both 24 and 48 h (Figure 7c). Cellular viability of the
AAV5-GFP treated CCMs was also greater than 95% at both 24 and 48 h (results not shown).
4. DISCUSSION
The results of this study demonstrate that AAV5 is a safe and effective vector for gene
therapy in CCFs and CCMs in vitro. To our knowledge this is the first study to test the efficacy
of an AAV5 vector for canine corneal gene therapy. The immunogenicity of AAV5 in the
canine cornea is currently not known. This in vitro study demonstrated no significant cell death
or phenotypic change associated with AAV5 gene therapy on CCFs and CCMs. Our previous
studies have demonstrated AAV to be nonpathogenic and safe for corneal gene therapy in vitro
in equine corneal fibroblasts, as well as in vivo in both the rabbit and mouse without causing
significant immune reactions.1, 14, 21, 25 However, future canine studies are warranted in order to
establish the safety and immunogenicity of AAV5 in vivo. Investigation of the effects of varying
viral concentrations, administration time, and gene delivery systems are warranted.
Use of an in vitro model allows for experimentation within a controlled environment
without risk to a clinical patient until safety parameters have been established. The in vitro
transduction efficiency of AAV5 determined for various cell types including lung epithelium,
skeletal muscle, gastrointestinal cells, and corneal fibroblasts is similar to reported in vivo
transduction efficiencies.73-75
Recently our lab demonstrated a higher level of gene transduction
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in vivo than previously published transgene expression in vitro with the topical application of
serotypes AAV9 and AAV8.16, 21
Therefore, the transduction efficiency percentage obtained
from this in vitro study concerning the efficacy of AAV5 as a vector for canine corneal gene
therapy may, in fact, underestimate the potential transgene expression that we can expect in vivo.
The discrepancies reported in the literature among transgene expression in vitro and in vivo
highlights the importance of validating in vitro findings with in vivo studies.16
The main cell type that plays a crucial role in fibrosis and scarring is the myofibroblast
which is derived from activated fibroblasts. The present study aimed to investigate the potential
use of gene therapy in CCFs as a model of the pre-fibrotic state as well as in CCMs, as a model
of corneal fibrosis. We found a significant decrease in transduction efficiency in myofibroblasts
compared to corneal fibroblasts. This difference in transduction efficiency of AAV5 between the
two cell types may suggest that higher doses of vector are required for delivering therapeutic
genes into fibrotic tissue. However, it may also be due to specific cell tropism of the viral
vector. Different AAV serotypes have varying shell proteins and therefore differ in their
capability of binding to and thus transfecting different host cells.21 Future studies are underway
to investigate different AAV serotypes in order to determine if a particular serotype demonstrates
specific tropism for canine myofibroblasts. This noted difference in transduction efficiency may
also signify the importance of therapeutic strategies aimed at initial prevention of corneal fibrosis
versus treatment of established fibrosis.
Furthering our understanding of the cascade of events that occurs in canine corneal
wound healing is critical to developing medical therapies that aim to prevent or reverse corneal
fibrosis. Corneal gene therapy makes use of a vector system to transfer a gene into the cornea so
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that a transgenic protein will be expressed to modulate congenital or acquired disease. The
protein may be structural (e.g. collagen), or functionally active (e.g. an enzyme, cytokine or
growth factor).52
Interfering with the activation of signaling transduction molecules using gene
transfer could be an important strategy to prevent or treat the undesirable outcomes of tissue
injury resulting in vision loss.15
Fibrotic diseases are characterized by the presence of
myofibroblasts, excessive accumulation of disorganized extracellular matrix, and subsequent
tissue contraction, all of which are modulated by a multitude of cytokines. Of those cytokines
TGFβ has been found to play a critical role in the development of corneal fibrosis following
injury by facilitating the trans- differentiation of keratocytes to myofibroblasts.
15
Decorin, a
small proteoglycan, which is a natural inhibitor of TGFβ has been successfully delivered to the
cornea by the use of gene therapy. Mohan et al. recently showed that decorin gene transfection
inhibits TGFβ-driven elevated expression of profibrogenic genes and transformation of corneal
fibroblasts to myofibroblasts in human corneal fibroblast cultures.59
The ability of gene therapy
to prevent corneal scarring in experimental animals has been demonstrated in several other
recent studies as well. Seitz et al. evaluated the use of retroviral vector-mediated genetic
transduction of keratocytes in vivo modulating keratocyte proliferation and decreasing corneal
scarring.61
They employed the use of a suicide gene and pro-drug combination of the herpes
simplex virus thymidine kinase (HSVtk) gene with ganciclovir. HSVtk triggers cell death in
proliferating keratocytes by converting ganciclovir to a toxic metabolite. This combination gene
therapy reduced corneal scarring 10 and 21 days post administration. Galiacy et al. utilized an
AAV vector expressing a fibril collagenase, matrix metalloproteinase 14 (MMP14), in a murine
model of corneal scarring.76
They observed that MMP14 gene delivery reduced corneal opacity
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and expression of several major genes involved in corneal scarring. These findings are
encouraging with regards to new developments for the prevention and treatment of corneal
fibrosis.
To our knowledge, this study is the first to investigate the effect of gene transfer in the
canine cornea. Our results demonstrate that AAV5 is a safe and effective vector for canine
corneal gene therapy in this in vitro model. In vivo studies are warranted.
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CHAPTER 4:
EFFICACY AND SAFETY OF SUBEROYLANILIDE HYDROXAMIC
ACID (VORINOSTAT) IN THE TREATMENT OF CANINE CORNEAL
FIBROSIS
1. EXPERIMENTAL PURPOSE
The purpose of this project is to examine the safety and efficacy of suberoylanilide
hydroxamic acid (SAHA) to treat canine corneal fibrosis using an in-vitro model.
2. MATERIALS AND METHODS
Canine corneal fibroblast and myofibroblast cultu res
Corneal buttons were aseptically harvested from purpose-bred healthy research dogs
receiving no topical or systemic medications, euthanized for reasons unrelated to the present
study. Slit-lamp biomicroscopy was performed by an experienced veterinary ophthalmologist
(EAG) prior to euthanasia to ensure that all samples were harvested from dogs free of anterior
segment disease. Corneal biopsy samples were washed with minimal essential medium (MEM,
Gibco; Grand Island, NY, USA) and the epithelium and endothelium were removed by careful
manual scraping using a # 10 Bard Parker scalpel blade (BD, Franklin lakes, NJ, USA). Corneal
stroma was sectioned into four smaller explants and placed in 100 X 20 mm tissue culture plates
(BD BioSciences, Durham, NC, USA) containing MEM supplemented with 10% fetal bovine
serum (Gibco, NY, USA) then incubated in a humidified CO2 incubator (Thermo Scientific,
Asheville, NC, USA) at 37oC to produce canine corneal fibroblast (CCF) cultures. In
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approximately 3-10 days, primary fibroblasts began migrating from the stromal sub-sections. On
attaining ~90% confluence the explants were manually removed using rat-toothed forceps and
discarded. Confluent cells were trypsinized and cellular concentrations were calculated using
Countess automated cell counter (Invitrogen, Carlsbad, CA, USA) and replated in 100 mm tissue
culture dishes. Passages 1-3 of canine corneal fibroblasts (CCFs) were used for this experiment.
Seventy-percent confluent CCF cultures in 12-well plates were used for all experiments. To find
the optimal dose of SAHA, dose-dependent studies were first performed. Briefly, SAHA at
concentrations of 0.01%, 0.02%, 0.06%, and 0.12% (2 μL for 12 well plates) was applied in
serum-free MEM medium for 5 min. After 5 min, the cells were washed and re-plated with fresh
medium, and cellular viability was assessed (see cytotixicity studies). Following preliminary
dose finding studies, a concentration of 0.06% (1 μL of 25nM) SAHA was used for all
subsequent experiments. At a time point of 4 h after treatment with TGF-β1 CCFs were treated
with 0.06% SAHA in serum-free MEM medium with TGF-β1 for 5 minutes (group 1) and for 24
h (group 2).
TGF-β1 was used to transform CCFs into myofibroblasts as an in-vitro model of canine
corneal fibrosis. For myofibroblast generation, CCF cultures were exposed to 1 ng/ml TGF-β1
(PeproTech Inc, Rocky Hill, NJ, USA) under serum free conditions. Qualitatively the formation
of myofibroblasts was confirmed using immunohistochemical staining for α-smooth muscle actin
(αSMA), which is specific to myofibroblasts. Quantitative assessment of alterations in
myofibroblast formation due to drug treatment was evaluated using real-time PCR and
immunoblotting.
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Cytotoxi city Studies
Cellular viability was assessed using trypan blue assay (Sigma-Aldrich, St. Louis, MO,
USA). Cells were trypsinized 48 hours after initiation of treatment and mixed with 0.4% trypan-
blue solution (Invitrogen, Carlsbad, CA, USA). Cell counts were performed using an automated
cell counter (Invitrogen, Carlsbad, CA, USA) and cellular viability was calculated as a percent.
Light microscopy images were obtained to observe any phenotypic changes in cell
cultures after the application of SAHA. CCF cultures treated with SAHA were examined with a
Leica fluorescent microscope (Leica, Wetzlar, Germany) and photographed at 8 h, 24 h, and 48 h
after treatment.
Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling
(TUNEL) assay was used to detect cellular apoptosis due to SAHA treatment. Cells were fixed
with 4% paraformaldehyde at room temperature for 15-20 min and then washed with phosphate
buffered saline. A fluorescence-based TUNEL assay was used according to the manufacturer's
instructions using ApopTag apoptosis detection kit (Chemicon international, Temecula, CA,
USA). Photographs were obtained using a fluorescent microscope (Leica, Wetzlar, Germany)
equipped with a digital camera (SpotCam RT KE, Diagnostic Instruments Inc., Sterling Heights,
MI, USA).
Immunocytochemistry
Immunocytochemical analysis was performed to confirm cell type. Immunofluorescent
staining for F-actin, which stains cytoskeletal cellular structures, and for αSMA, a marker for
myofibroblasts, was performed using mouse monoclonal antibody for phalloidin and αSMA,
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respectively. At study end point cultures were washed twice with PBS and incubated at room
temperature with mouse monoclonal antibody for phalloidin (Invitrogen, Calsbad, CA, USA) or
αSMA (DAKO, Carpinteria, CA, USA) at a 1:500 dilution in 1X PBS for 90 minutes. After
incubation with the primary antibody, cells were washed twice with PBS, then incubated with
secondary antibody Alexa 488 or 594 goat anti-mouse IgG (Invitrogen, Carlsbad, CA, USA) at a
dilution of 1:500 for 1 h. Cells were mounted with Vectashield containing 4'-6'-Diamidino-2-
phenylindole (DAPI) (Vector laboratories, Inc., Burlingame, CA, USA) to allow visualization of
nuclei. Irrelevant isotype-matched primary antibody, secondary antibody alone, and tissue
sections from naïve eyes were used as negative controls for each immunocytochemistry
experiment. Immunostained cultures were examined and photographed with Leica fluorescent
microscope (Leica, Wetzlar, Germany) equipped with a CCD digital camera (SpotCam RT KE,
Diagnostic Instruments Inc., Sterling Heights, MI, USA).
Immunoblotting
Cells were washed with ice-cold PBS and lysed directly on plates using RIPA protein
lysis buffer containing protease inhibitor cocktail (Roche Applied Sciences, Indianapolis, IN,
USA). Samples were suspended in Laemmli's denaturing sample buffer (30μl) containing β-
mercaptoethanol, vortexed for 1 min, centrifuged for 5 min at 10,000 x g, and then boiled for
70oC for 10 min. Protein samples were resolved by 4%-10% SDS-PAGE and transferred to a
0.45-μm pore sized PVDF membrane (Invitrogen, San Diego, CA). αSMA monoclonal antibody
was purchased from DAKO (Carpinteria, CA, USA) and GAPDH antibodies from Santa Cruz
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Biotechnology Inc (Santa Cruz, CA, USA). Anti-mouse or goat-AP antibodies were obtained
from Santa Cruz Biotech (Santa Cruz, CA, USA).
Quantif ication using r eal-time PCR
Total RNA from CCF cultures treated with SAHA was extracted using RNeasy kit
(Qiagen Inc., Valencia, CA, USA) and reverse-transcribed to cDNA following manufacturer's
instructions (Promega, Madison, WI, USA). Real-time PCR (RT PCR)was performed using iQ5
real-time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA). A 50 µL reaction
mixture containing 2 µL cDNA (250 ng), 2 µL forward (200nM), 2 µL reverse primer (200nM),
and 25 µL 2X iQ SYBR green super mix (Bio-Rad Laboratories) was run at universal cycle
(95oC for 3 min, 40 cycles at 95
oC for 30 sec, and 60
oC for 60 sec) following manufacturer's
instructions. For αSMA, forward primer sequence TGGGTGACGAAGCACAGAGC and
reverse primer sequence CTTCAGGGGCAACACGAAGC were used. House keeping gene β-
actin forward primer sequence was CGGCTACAGCTTCACCACCA and reverse primer
sequence was CGGGCAGCTCGTAGCTCTTC. Cycle threshold (CT) was used to detect
increases in signal associated with an exponential growth of PCR product during the log-linear
phase. Relative gene expression was calculated using the following formula: 2-ΔΔC
T, where CT is
a PCR cycle number at which the fluorescence meets the threshold in the amplification plot and
ΔCT is a subtraction product of target and housekeeping genes CT values. The 2-ΔΔC
T is a
method for determining relative target mRNA quantity in samples. Amplification efficiency for
all used templates was validated by insuring that the difference between linear slopes for all
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templates <0.1. Three independent reactions were performed and the average (± standard error of
the mean or SEM) results were calculated.
Quant if ication and statistical analysis
Immunocytochemistry data quantification was performed by counting αSMA and DAPI-
positive cells in 10 randomly selected non-overlapping areas of fluorescent cultures at 100X
magnification field under Leica Fluorescent microscope (Leica, Wetzler, Germany). Results are
expressed as mean ± standard error of the mean (SEM). Statistical analysis was performed using
two-way analysis of variance (ANOVA) followed by Bonferroni test for comparisons test for
cell toxicity assay. The RT PCR results were analyzed using one-way ANOVA followed by
Tukey's multiple comparison tests. A P-value of less than 0.05 was considered significant.
Immunoblotting data were analyzed using J 1.38 X image analysis software (NIH, Bethesda,
MD, USA)
3. RESULTS
SAHA safety
Cellular viability of CCFs after SAHA treatment was determined by trypan blue
exclusion assay (Fig. 8). A dose of 0.06% SAHA treatment was the highest concentration tested
that did not significantly alter cellular viability. Toxicity and safety of SAHA treatment was
evaluated by comparing phenotype and cell death in SAHA-treated and untreated CCFs. Light
microscopy images of untreated and 0.06% SAHA-treated CCFs were obtained to observe any
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phenotypic cellular changes. SAHA 0.06% treatment for 5 min (group 1) or 24 h (group 2) 4 h
after TGFβ1 treatment did not alter CCF phenotype (Fig. 9) compared to corresponding controls.
Similar results were seen 48 h after treatment in both groups (data not shown).
The TUNEL assay, which predominantly detects apoptosis and to a lesser extent necrosis,
demonstrated the safety of SAHA in this in vitro model. No detectable difference in cellular
apoptosis was observed between 0.06% SAHA treated (group 1 or 2) cells and untreated controls
(Fig. 10).
Immunocytochemistry and αSMA quantification
Results of Immunocytochemistry staining of myofibroblast transformation is shown in
Figure 11. Approximately 3-5% myofibroblast cells in corneal fibroblast cultures (Fig. 11a) is
normal and unavoidable.77
Treatment of CCFs with TGFβ1 stimulated transdifferentiation of the
majority of fibroblasts into myofibroblasts (Fig. 11b), as depicted by a significant increase in
αSMA staining. Both group 1 (5 min) and group 2 (24 hr) 0.06% SAHA treatments showed a
significant decrease in αSMA immunostaining when compared to the TGFβ1 treated control
groups (Fig 11c and 11d).
Quantification of αSMA immunostaining is shown in Figure 12. TGFβ1 treatment of
CCFs produced a significantly higher level of αSMA staining (89 ± 2.2%; p<0.01) when
compared to the untreated control group (8 ± 2%). Treatment with 0.06% SAHA significantly
decreased TGFβ1-induced myofibroblast production in both group 1 (26 ± 7.4%, p<0.01) and
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group 2 (23 ± 1.6%, p<0.01). The comparison of αSMA immuno-detection data between SAHA
treatment groups 1 and 2 showed no significant difference in αSMA levels.
αSMA real -time PCR and western bl ot
The relative expression of αSMA in the untreated control group, the TGFβ1 treatment
group, and 5 min (group 1) and 24 h (group 2) SAHA treatment groups was quantified using
real-time PCR (Fig. 13). In both SAHA treatment groups 1 and 2 a significant decrease in
αSMA expression was detected when compared to the TGFβ1 treatment group. The percentage
of αSMA mRNA level was significantly lower in both SAHA treatment groups 1 and 2
compared to control. αSMA mRNA levels were not significantly different between SAHA
treatment groups 1 and 2.
Western blot analysis of αSMA protein (Fig. 14) was consistent with our quantitative
mRNA and immunocytochemistry findings. Treatment of CCFs with 0.06% SAHA effectively
suppressed αSMA expression in both treatment groups 1 and 2.
4. DISCUSSION
Corneal wound healing involves numerous cellular mechanisms including corneal
epithelial and keratocyte migration, proliferation, apoptosis, matrix remodeling, and
transdifferentiation of keratocytes to fibroblasts and myofibroblasts.26, 78, 79
This process is
regulated by several factors including various growth factors, cytokines, and chemokines.26, 80
79,
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81 Of the various cytokines involved in the formation of corneal fibrosis, TGFβ has been found
to play a pivotal role in initiating this transdifferentiation. The development of corneal fibrosis is
greatly dependent on the generation of myofibroblasts.4, 82, 83
Myofibroblasts are highly
contractile cells with decreased intracellular crystallin production resulting in reduced corneal
transparency.
Currently, topical mitomycin C (MMC), an alkylating chemotherapeutic agent, is
commonly used in physician ophthalmology to prevent laser-induced corneal haze. MMC blocks
keratocyte proliferation. Recently our lab reported that MMC effectively reduced the formation
of myofibroblasts in the equine
77
and canine cornea
84
in vitro and in rabbit cornea in vivo.
85
However, MMC application is associated with several complications including corneal
endothelial damage, abnormal wound healing, limbal and scleral necrosis, loss of keratocytes,
and the inhibition of keratocyte repopulation.85, 86
As a class of drugs, HDACi alter gene expression profiles at the epigenetic level and
change protein function by inhibiting the activity of HDAC. HDACi were originally developed
for their anti-proliferative properties in transformed cells.87, 88 They are currently being
extensively investigated in cancer research for their anti-angiogenic properties and their ability to
induce apoptosis and cell cycle arrest in malignant cells.89
Additionally, HDACi also prevent
pro-inflammatory cytokine production and have demonstrated anti-inflammatory activities in
animal models of inflammatory bowel diseases, multiple sclerosis and SLE.90-93
The therapeutic
potential of HDACi as antifibrotic agents has been recently reported in lung and cardiac
fibrosis.94, 95
In this study we demonstrated that 0.06% SAHA, an HDACi, significantly
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decreased the amount of myofibroblast formation without significant cellular toxicity using an
established canine corneal in vitro model.
The precise mechanism through which SAHA inhibits TGFβ1 stimulated myofibroblast
formation remains unclear. It is reported that the inhibition of HDAC4, HDAC6, and HDAC8
impair TGFβ1 induced αSMA expression.96
The inhibition of HDAC4 has been found to prevent
morphological changes associated with TGFβ1 stimulation.96
Down-regulation of HDAC4
stimulated the expression two endogenous repressors of the TGFβ signaling pathway but did not
stimulate inhibitory Smad7.96
Therefore HDAC4 may be a potential specific target for treating
fibrotic disease.
Studies specifically aimed at examining the use of SAHA in fibrotic disease are limited.
A recent report investigated the effects of SAHA on human lung fibroblast cultures as a
therapeutic potential for pulmonary fibrosis. That study showed an inhibition of TGFβ1 induced
transdifferentiation of fibroblasts to myofibroblasts, demonstrated by the suppression of αSMA
expression. Furthermore, SAHA treatment reduced TGFβ1 expression and collagen I deposition
in the tested cell type without causing apoptosis.94
A significant reduction in αSMA expression
due to SAHA treatment was consistent with our findings reported here.
Inflammation typically precedes fibrosis in fibro-proliferative diseases, thus the anti-
inflammatory properties of SAHA may help to prevent fibrosis. Known anti-rheumatic activities
of SAHA include growth arrest of rheumatoid arthritis synovial fibroblasts, suppression of pro-
inflammatory cytokines and nitrous oxide (NO) release, and down-regulation of angiogenesis
and MMPs.97
SAHA has also been reported to reduce circulating levels of multiple pro-
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inflammatory cytokines in a rat model of hypertensive cardiomyopathy.95
Although we did not
specifically evaluate fibroblast proliferation in this study, future studies may involve the use of
an MTT ((3-4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay to assess
fibroblast proliferation as well as investigation of underlying molecular mechanisms of SAHA-
mediated anti-fibrosis in the canine cornea.
To the authors’ knowledge, this is the first study to evaluate the use of an HDACi,
specifically SAHA, to prevent corneal fibrosis in a canine in vitro model. Our results suggest
that SAHA may be a potent agent to treat fibrosis in the canine cornea. In vivo studies are
warranted.
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APPENDIX
Vector Immunogenicity
Long-term
expression
Transduction of
Dividing (D) or
Non-dividing (ND)
cells
AV Moderate-severe No D
AAV No-mild Yes D & ND
Lentivirus Mild-moderate No D & ND
Disabled
Lentivirus Mild-moderate Yes D & ND
Retrovirus Mild-severe No D
Table 1: Characteristics of viral vectors used in corneal gene therapy.
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Figure 1. Representative immunocytochemistry images demonstrating phalloidin staining for F-
actin (shown in red) in canine corneal fibroblasts (a) and myofibroblasts (b). Cellular nuclei are
stained blue with DAPI. Myofibroblasts (b) show significantly higher amount of F-actin staining
compared to and fibroblasts (a) revealing the levels of fibrosis.
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Figure 2. Representative images demonstrating delivered GFP staining in CCFs. AAV5-
transduced CCFs (b) show significantly higher number of GFP-positive cells compared to the
untreated control cell population (a). Dual staining was performed to detect αSMA (shown in
red), a marker for myofibroblasts.
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Figure 3. Representative images demonstrating delivered GFP staining in CCMs (b). AAV5-
transduced CCMs (b) show significantly higher number of GFP-positive cells compared to the
untreated control cell population (a). Dual staining was performed to detect αSMA (shown in
red), a marker for myofibroblasts.
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Figure 4. Quantification of GFP positive cells showing the amount of gene delivery by AAV5
into CCFs as well as CCMs. In both cell populations there is significant transduction of AAV5-
GFP (grey bar) compared to the untreated controls (black bar). However, the percentage of
GFP-positive cells is significantly lower in CCMs compared to CCFs. Error bars indicate
standard error. * p <0.01 versus control; Ψ p<0.01 versus AAV5-GFP CCFs.
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Figure 5. Quantitative PCR measurements of AAV5-delivered GFP in CCFs and CCMs. Both
cell populations showed significant transduction of AAV5-GFP (crossed bar) compared to the
untreated controls (black solid bar). However, the levels of GFP-mRNA were significantly lower
in CCMs compared to CCFs. Error bars indicate standard error. * p <0.01 versus control; Ψ
p<0.01 versus AAV5-GFP CCFs.
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Figure 6. Phase-contrast microscopic images of CCF (a) and CCM (b) cultures taken 48 h after
AAV transduction showed that AAV5 viral vector application did not alter the morphology of
the fibroblast and myofibroblast culture population. Similar results were seen at 24 h.
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Figure 7. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay
demonstrating apoptotic cells (pink staining) in CCF cultures transduced with AAV5-GFP.
AAV5-GFP treated cells (b) demonstrated a comparable number of TUNEL-positive cells (white
arrows) to untreated controls (a). Similar results were seen for CCMs. Nuclei are stained blue
with DAPI. Trypan Blue exclusion data (c) shows the percent viability of CCFs 24 h and 48 h
following AAV transduction compared to controls. Percent cellular viability slightly decreased
(results not significant) in AAV5-GFP treated CCFs (black bars) and CCMs (results not shown)
compared to the untreated controls (white bars). Error bars indicate standard error.
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Figure 8:
A bar graph showing the cellular viability as determined by trypan blue assay in order to
demonstrate the dose dependent effect of SAHA on CCFs. Doses of 0.06% SAHA or less did
not alter cellular viability. However, 0.12% SAHA demonstrated mild cellular toxicity.
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Figure 9:
A composite image showing phase-contrast microscopy images of CCFs at 5 minutes and 24
hours. No changes in CCF morphology were observed in group 1 (b) or group 2 (d) 0.06%
SAHA treatment, when compared with their respective control groups (a & c). Bar = 10 μm
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Figure 10:
Representative images of TUNEL assay results detecting apoptosis of CCFs in the untreated
control group (a) and in CCFs treated with 0.06% SAHA for 24 h (b). No detectable differences
in cellular apoptosis were observed between treated and control groups.
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Figure 11:
Immunocytochemical staining using αSMA antibody (green). Detection of ~ 3-5%
myofibroblast cells in corneal fibroblast cultures (a) is normal and unavoidable.25
Treatment of
CCFs with TGFβ1 showed significant increase in αSMA staining (b). Both group 1 (c) and
group 2 (d) 0.06% SAHA treatment groups showed a significant decrease in αSMA
immunostaining when compared to the TGFβ1 treated control group (b).
Group 1 (5 min)
Control TGFβ
Group 2 (24 hrs)
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Figure 12:
Bar graphs showing αSMA immunostaining quantification. TGFβ1 treatment of CCFs produced
a significantly higher level of αSMA staining when compared to the untreated control group.
Treatment with 0.06% SAHA significantly decreased TGFβ1-induced myofibroblast production
in both the brief and extended treatment groups. # denotes p<0.01 (control vs TGFβ1) and *
denotes p<0.01 (TGFβ1 vs SAHA treatment groups). No significant difference in αSMA levels
were found between the brief and extended SAHA treatment groups.
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Figure 13:
Bar graphs showing quantitative real-time PCR results demonstrating the relative expression of
αSMA . In both group 1 (5 min) and group 2 (24 h) SAHA treatment groups there was a
significant decrease in αSMA expression compared to the TGFβ1 treatment group. * denotes
p<0.01 (control vs TGFβ1) and ** denotes p<0.01 (TGFβ1 vs SAHA treatment groups 1 & 2).
αSMA mRNA levels were not significantly different between the brief and extended SAHA
treatment groups.
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Figure 14:
Western blot analysis of αSMA protein. Treatment of CCFs with 0.06% SAHA effectively
suppressed αSMA expression in both treatment groups 1 (5 min)& 2 (24 h).
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