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The roles of type VII collagen in wound healing and scar reduction
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The roles of type VII collagen in wound healing and scar reduction
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Content
THE ROLES OF TYPE VII COLLAGEN IN WOUND
HEALING AND SCAR REDUCTION
by
Xinyi Wang
A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(PATHOBIOLOGY)
December 2014
Copyright 2014 Xinyi Wang
i
Epigraph
I can be changed by what happens to me. But I refuse to be reduced by it.
--Maya Angelou
ii
Dedication
I dedicate this doctorial dissertation to my family, for their unspeakable grace,
unwavering support and love. I would not have been through this long but rewarding
journey without them.
To my mother, Ye Li, a competent and brave woman: Thank you for always being
there for me.
To my late grandfather, Tingying Li, and my late father, Junming Wang: I am
sorry for not being there for you.
I also would love to dedicate this work to all RDEB patients’ families, especially
the parents, I witness your unbelievable love and patience; to all RDEB patients, hope
you can be relieved from the devastating disease soon. Thank you for showing me the
shining points in humanity.
iii
Acknowledgements
My gratitude to those who have helped and encouraged me all the way in this
fruitful journey cannot be fully expressed in two pages. I would like to take this
opportunity to acknowledge the following people.
Dr. Mei Chen, my mentor. This highly motivated, disciplined and successful
scientist has taught me from the simplest pipetting techniques all the way to writing my
own PhD dissertation. Her dedication and passion in science has garnered my utmost
respect. I thank her for her commitment, tremendous support and belief in me. She
paved a solid and bright ground for my career.
Dr. Wei Li, a great teacher and passionate scientist. I truly appreciate all the trust
and opportunities he has given me. He makes learning science interesting and
masterfully. The finer details he emphasized in science make all the difference between
success and failure.
Dr. David Woodley, an experienced and trusted adviser and pioneer. It was his
vision and leadership that started this great bench to bedside laboratory.
My Ph.D dissertation and guidance committees, Dr. Florence Hofman, Dr.
Cheng-Ming Chuong and Dr. Baruch Frenkel, and Ph.D guidance committees, Dr. Vijay
Kalra and Dr. Louis Dubeau. Their insightful guidance, enormous support and generous
encouragements are indispensable.
My long time and current colleagues, Dr. Yingping Hou and Mr. Jon Cogan as
well as my former colleagues Ms. Pubali Bandyopadhyay Mukherjee and Dr. Jennifer
Remington. They have created a great working atmosphere in Dr. Chen’s laboratory, and
I thank them for teaching and helping me in every way possible. It has been a wonderful
experience to work with them.
The medical/undergraduate students who have worked in Dr. Chen’s laboratory,
Dr. Pedram Ghasri, Dr. Mahsa Smith, Mr. Michael Khalili, Dr. Brian Hwang, Dr.Erica
Cua, Mr. Robert Gao, Mr. Timothy Wu, Mr. Cyrus Haghighian, Ms. Kaity Yim and Dr.
iv
Andrew Lin, I appreciate their dedication and time during the period they worked in the
laboratory. Working with these intelligent and goal-driven students have made my life
happier.
It has been a wonderful time in the big family, the Department of Pathology at
USC. Our graduate advisor, Ms. Lisa Doumak has always been there to provide help and
solutions to graduate students without fail.
The graduated students in Dr. Li’s laboratory, Dr. Shengxi Guan, Dr. Jack Cheng,
Dr. Fred Tsen, and Dr. Divya Sahu, I have always valued their advice and suggestions.
My scientific collaborators, Dr. Douglas Keene, Dr. Jouni Uitto, Ms. Lillian
Young, and Ms. Michelle Macveigh, for their work and help made this thesis happen.
Mr. Christian Lise and Ms. Vivian Ning, I truly appreciate their help and patience
to proofread my thesis.
Ms. Kelly Zhou, Dr. Meng Xu, Dr. Xiuqing Li and Ms. Sarah Vargas, I am so
grateful for they knowing all about me and still love me. To have friends like them also
encourage me to keep pursuing better things in my life.
Last but definitely not the least, my lovely family members, especially my mother
Ye Li, makes everything possible in my life; grandma Shifen Wang, the years I spent
with you and grandpa have been the best memories in my life.
For those I do not name individually, please accept my generalized appreciation.
v
List of Tables
4.1 Summary of C7 expression and AFs in RDEB patients’ skin and
anti-C7 antibodies in the blood
81
vi
List of Figures
1.1 The domain organization of a C7 alpha chain and how C7 molecules
assemble into AFs
14
2.1 Clinical, histological, and immunological presentation of DEB mice 30
2.2 Presence of C7 in DEB mice following protein injection 31
2.3 Absence of C7 in blood stream and internal organs 31
2.4 Immunoelectron microscopy of mouse skin injected with human rC7 32
2.5 Improved survivals of DEB mice by C7 injections 32
2.6 Anti-C7 IgG productions in DEB mice after protein therapy 33
2.7 Anti-C7 IgG neither bound to the skin directly nor inhibited further
BMZ incorporation of newly injected rC7
34
2.8 Suppression of anti-C7 antibody production by Mr1 treatment 35
3.1 Topically applied rC7 stably incorporated in the regenerated BMZ in the
mouse skin
53
3.2 Topical application of rC7 promoted wound healing 54
3.3 Topical application of NC1 did not promote wound healing 57
3.4 C7 inhibited the contraction of collagen lattices in vitro and reduces the
presence of myofibroblasts, CTGF expression, and collagen deposition
in vivo.
57
3.5 Topically applied rC7 promoted wound closure of human skin 59
3.6 Topical application of rC7 incorporated into the BMZ of RDEB mouse
skin grafts and forms AFs in vivo
59
4.1 The profiling of TGF β pathway markers in RDEB patients 82
4.2 Lentiviral vector mediated gene transfer of C7 to RDEB fibroblasts
reduces the expressions of TGF β pathway and fibrosis markers.
85
4.3 Lentiviral vector mediated gene transfer of C7 to RDEB keratinocytes
reduces the expression of TGF β pathway markers.
86
4.4 Supplementing C7 or NC1 to RDEB fibroblasts decreases the
expression of TGF β1 induced the activation of p-Smad 2/3
87
4.5 siRNA mediated knockdown of C7 in normal fibroblasts increases the
expression of TGF-β pathway and fibrosis markers
88
4.6 TGF-β pan specific antibody reduces the expression of TGF-β1 and α-
SMA in RDEB fibroblasts
88
4.7 TGF-β R-I inhibitor decreases the expression of α-SMA in RDEB
fibroblasts
89
4.8 siRNA mediated knockdown of Smad-2 or Smad-3 in RDEB fibroblasts
decreases the expression of TGF-β pathway and fibrosis markers
89
4.9 The schematic model of how C7 responsible for altered TGF-β fibrosis
signaling and hyper-contraction activity in RDEB fibroblasts
90
vii
Abbreviations
AF
Anchoring fibrils
ALK5
Anaplastic lymphoma kinase
BMZ
Basement membrane zone
BSA
Bovine serum albumin
BSLE
Bullous systemic lupus erythematosus
C1
Type I collagen
C4
Type IV collagen
C7
Type VII collagen
CMP
Cartilage matrix protein
cSCC
Cutaneous squamous cell carcinoma
CTGF
Connect tissue growth factor
DDEB
Dominant dystrophic epidermolysis
bullosa
DEB
Dystrophic epidermolysis bullosa
DMEM
Dulbecco's modified essential medium
EB
Epidermolysis bullosa
EBA
Epidermolysis bullosa acquisite
EBS
Epidermolysis bullosa simplex
ECM
Extracellular matrix
ELISA
Enzyme-linked immunosorbent assay
FDA
Food and Drug Administration
FN-III
Fibronectin type III like domains
FPCL
Fibroblasts populated collagen lattice
assay
H&E
Hematoxylin & Eosin
ID
Intradermal
Ig
Immunoglobulin
IHC
Immunohistochemistry
IV
Intravenous
JEB
Junctional epidermolysis bullosa
MR1
Major histocompatibility complex related
protein-1
NC1
Amino-terminal, non-collagenous
domain 1
NC2
Carboxyl-terminal, non-collagenous
domain 2
PCNA
Proliferate cell nuvlear antigen
PDGF
Platelets-derived growth factor
viii
RDEB
Recessive dystrophic epidermolysis
bullosa
siRNA
small interfering RNA
TGF β
Transforming growth factor beta
TGF-β R
Transforming growth factor beta receptor
WT
Wild type
α-SMA
Alpha smooth muscle actin
ix
Table of Contents
Epigraph i
Dedication ii
Acknowledgements iii
List of Tables v
List of Figures vi
Abbreviations vii
Chapter I: Introduction
1.1 Structure and Function of Type VII Collagen 1
1.2 Type VII Collagen related Diseases 2
1.3 Skin Wound Healing and Fibrosis 6
References 11
Figure 14
Chapter II: Intradermal Injection of Recombinant Human Type VII Collagen Corrects
the Disease Phenotype in a Murine Model of Dystrophic Epidermolysis Bullosa
2.1 Summary 15
2.2 Results 16
2.3 Discussion 20
2.4 Materials and Methods 24
References 28
Figures 30
Chapter III: Topical Application of Recombinant Type VII Collagen Incorporates Into
the Dermal-Epidermal Junction and Promotes Wound Closure
3.1 Summary 36
3.2 Results 37
3.3 Discussion 42
3.4 Materials and Methods 47
References 51
Figures 53
Chapter IV: The Absence of Functional C7 in RDEB Causes Faulty Regulation of TGF
β and Profound Skin Scarring
4.1 Summary 61
4.2 Results 62
4.3 Discussion 68
4.4 Materials and Methods 73
References 79
Figures 81
1
Chapter I
Introduction
1.1 Structure and Function of Type VII Collagen
Type VII collagen (C7) was first identified and described as “ a collagen with a
triple helical domain about 1.5 times the length of type I collagen” in 1983 [1]. In
humans, C7 is encoded by the COL7A1 gene, which is located on the short arm of
chromosome 3 at position 21.1 [2]. The entire gene is 32kb including 118 exons and
relatively small introns, encoding a 8.9kb mRNA [3]. Its cDNA has a 8833 nucleotides
open reading frame encoding 2,944 amino acids [4]. A C7 molecule is composed of
three identical α chains stabilized by interchain disulfide bonds. Each α chain consists of
a large 145 kDa amino-terminal, non-collagenous domain (NC1) and a small 34 kDa
carboxyl-terminal non-collagenous domain (NC2) [5-7]. These two domains are
connected by a 145 kDa triple helical collagenous domain, which is characterized by
repeating Gly-X-Y amino acid sequences. In addition, there is a 39 amino acid non-
collagenous hinge region in the middle of the triple helical domain, which can be
digested by pepsin. Within the extracellular space, the C7 molecules form antiparallel,
tail-to-tail dimers, and the overlapped NC2 parts are removed during the proteolytic
process [7-10]. Then the dimers aggregate to form a structure called anchoring fibrils
(AFs) providing the stability of the epidermal and dermal adhesion. AFs are considered
as the structures connecting the lamina densa and papillary dermis. Papillary dermis is
located on the dermal side of the BMZ beneath the stratified squamous epithelium and
composed of two matrix layers: lamina lucida and lamina densa [11]. The schematic in
Figure 1.1 shows the domain organization of a C7 α chain and how C7 molecules
assemble into AFs. NC1 domain has multiple sub-modules that are homologous to some
adhesive proteins, including cartilage matrix protein (CMP) on the amino-terminus, next
is fibronectin type III-like (FN-III) domains, and von Willebrand factor on the carboxyl
terminus. The high affinity of NC1 domain with fundamental extracellular matrix (ECM)
and basement membrane zone (BMZ) proteins, collagen IV (C4), laminin 5, laminin 6,
fibronectin and collagen I (C1) provides the function of stabilization [12, 13]. NC2
2
domain with a short adjacent triple helical sub-domain is responsible for initiating triple
helix assembly and antiparallel dimer formation [9]. Both NC1 and NC2 domains
contain antigenic epitopes, which can be use to detect anti C7 autoantibodies in human
[14].
C7 is synthesized in skin keratinocytes and fibroblasts, and is expressed in skin,
oral mucosa, and cornea [12]. As a structural protein, the C7 gene structure and protein
sequence are highly conserved. For example, comparing the mouse C7 to human C7
shows 84.7% homology and 90.4% identity at the gene and protein level, respectively
[15].
1.2 Type VII Collagen Related Diseases
There are two types of bullous diseases related to C7: dystrophic epidermolysis
bullosa (DEB), which is due to mutation in gene encoding for C7, and epidermolysis
bullosa acquisite (EBA), which is an acquired autoimmune disease in which patients
develop autoantibodies to C7.
DEB is a subtype of a group of rare inherited connective tissue diseases,
epidermolysis bullosa (EB). Through minor traumas, EB could cause blisters in fragile
skin and mucosa. Based on the distinct molecular mechanisms, ultrastructural,
immunohisochemical and phenotypic findings, EB has been defined into three major
subtypes, epidermolysis bullosa simplex (EBS), junctional epidermolysis bullosa (JEB),
and DEB [16].
C7 gene is compact and complex. Over 700 distinct mutations have been reported
in the COL7A1 gene, including missense, frameshift, insertion, deletion, and nonsense
changes [3, 17]. The multitudinal series of mutations in the gene encoding C7 cause
either absence or modification of AFs, resulting in a broad range of phenotypic severity
observed in DEB patients’ skin and mucosa [18]. Minor frictions in daily life could
cause large blisters in the lamina densa. DEB patients have been described as butterfly
children because of the ultra-fragility of their skin. According to the inherited traits, DEB
is divided into autosomal dominant and autosomal recessive. When comparing dominant
3
dystrophic epidermolysis bullosa (DDEB) to recessive dystrophic epidermolysis bullosa
(RDEB), RDEB has absent or reduced AFs, while DDEB has only reduced AFs [19].
In an updated EB diagnosis and classification report, RDEB has four subtypes
according to the general clinical obvious severity. Listed from most severe to least
severe: RDEB, generalized, severe; RDEB, generalized, intermediate; RDEB, localized;
and RDEB all other subtypes [16].
Throughout their lives, RDEB patients have consistent open wounds and erosions
caused by inherited skin fragility. It is unavoidable to prevent the healed wounds from
generating redundant scars. These scars will cause many complications for RDEB
patients, including fibrotic fusion of digits, joints contractures of limbs, the
conglutination of lips, fixed tongue, the stenosis and synechia of esophagus, poor
dentition and nutritional deficiencies.
Besides all the mentioned devastating conditions, RDEB patients face
complications from another fatal consequence: cutaneous squamous cell carcinoma
(cSCC) [20]. cSCC is a common skin cancer originating from malignant proliferation of
epidermal keratinocytes; it has approximately 95 percent chance of recurrence within five
years. cSCC tumors are easy to spot and treat at early stages; however, they metastasize
frequently and aggressively, leading to a high mortality rate of up to 80 percent by age
50. Moreover, the metastasis rate of cSCC in RDEB patients is higher than other cSCC
patients with different causes, such as UV light, albinism and organ transplantations.
Treatment options for cSCC include radiation therapy, chemotherapy and amputation.
Nevertheless, these treatments have not shown to be successful in improving the cSCC
survival rate in RDEB patients. The reason for RDEB cSCC to be so malignant
compared to non-RDEB cSCC is still unclear [21, 22].
Two RDEB genetically modified and immune-competent murine models have
been broadly applied in research studies. Dr. Uitto’s lab generated a RDEB murine
model by inactivating the COL7A1 gene. C7 null mice have no C7 at the BMZ of their
skin. Consequently, they entirely lack ultra-structurally recognizable AFs, and exhibit
large blisters and erosions, and most likely die within a week after birth due to the
4
complications of the extensive blistering. Thus, these C7 null mice mimicked the genetic,
ultra-structural and clinical features of the most severe RDEB patients. However, due to
early demise, the time frame for researchers to work on is not sufficient. Moreover, it is
not suited for RDEB wound healing projects. Instead, we utilized RDEB skin
transplantation models, skin equivalent murine model, or normal murine model to
perform the experiments [23]. The other RDEB genetically modified murine model is
COL7A1 hypomorphic mouse generated from Dr. Tuderman’s lab. COL7A1
hypomorphic mice express C7 about 10% of physiological levels in wild type mouse and
replicate the main characteristics of human RDEB. These characteristics include
blistering, nail dystrophy, and mitten deformities of the extremities. Both models are
immune competent that can mount immune responses to the newly introduced C7 protein.
This provided us a great opportunity to assess the potential immune responses to the C7
based protein replacement therapy [24].
Since the early 21
st
century, researchers have developed ex vivo and in vivo
therapeutic approaches for DEB. In Ex vivo therapy, our laboratory showed that we
could transplant sheets of cultured keratinocytes autografts onto burn patients, to cover
their wound, reduce the loss of salt & water and inflammations. For RDEB patients, we
could culture their keratinocytes and gene corrected with a lentiviral vector gene
expressing C7, then transplant back onto the RDEB patients’ wounds. Khavari’s group
used the similar idea to apply on RDEB patients with a phi C31 integrase-based non-viral
transfer approach, achieving C7 restoration, AFs formation, and improved adherences
[25]. C7 has a remarkable ability to self-assemble in the extracellular space, so in the
past decade, Chen’s laboratory has established more direct therapies in vivo. We found
that gene corrected RDEB fibroblasts can be used to produce human recombinant C7
since they over-express C7 with gene correction. The phenotypes of corrected skin cells
also become more normalized in terms of growth, motility, and matrix adherent ability.
We utilized two mouse models to test our hypothesis; one is the athymic nude mouse
transplanted with human RDEB skin equivalents generated with RDEB keratinocytes and
fibroblasts, and the other one is normal mouse model. With these two models, we
intradermally injected (ID) (i) molecularly engineered RDEB fibroblasts synthesizing and
secreting large amounts of C7 protein. The C7 incorporated into the skin's BMZ and
5
formed anchoring fibril structures [26]. (ii) lentiviral vectors expressing C7. As early as
2004, Chen’s lab demonstrated that the lentiviral vector transduced dermal cells in both
models synthesized and exported C7 into the extracellular space. Moreover, C7
selectively adhered to and incorporated into the BMZ and formed anchoring fibril
structures. In the DEB skin equivalent model, the newly expressed C7 reversed the DEB
phenotype characterized by poor epidermal–dermal adherence and anchoring fibril
defects. C7 expression is stable at the BMZ, and a single lentiviral vector injection can
sustain for at least 3 months [27]. Intravenous injection (IV) delivery of C7 has also been
taken in consideration, since the severely affected RDEB patients are not only often have
multiple wounds all over the surface of body, but also have wounds in oral mucosa,
esophagus, and other inaccessible sites for ID treatments. Our Molecular Therapy paper
published in 2007, first demonstrated that IV injection of gene corrected DEB fibroblasts
accelerated healing the wounds made on murine and grafted human skin continuously
delivered C7 at the wound site [28]. All these therapies promoted new C7 expression,
functional AFs, improved adherence of dermal epidermal junction, and significantly
prolonged the life span of C7 knockout mice. Only a few RDEB clinical trials have been
reported. Wong et al. found that intradermal injections of allogeneic fibroblast into
RDEB patients would increase endogenous mutated C7 expressions in skin, causing less
skin bullae with improved dermal epidermal adherence [29]. Wagner and colleagues
exhibited that bone marrow/stem cell transplantation into RDEB patients led to improved
clinical outcomes [30]. Although the results are promising, neither therapy proves
consistently effective or with acceptable risk range.
EBA is the other rare, acquired and chronic bullous disease related to C7, in
which the patients have anti-C7 autoantibodies. The clinical features of EBA are similar
to the genetic forms of DEB. This includes blistering, erosions, skin fragility, milia, nail
loss and scars. EBA affects 1 person in 5 million, with no gender or racial preference.
The available medications and treatments include colchicine, dapsone, plasmapheresis,
photopheresis, infliximab, and intravenous immunoglobulin. However, none of these
medications can cure EBA completely. Chen’s lab and others have shown that NC1
domain of C7 has major antigenic epitopes, which can be the potential targets for EBA
autoantibodies. Moreover, more evidences have shown that antigenic epitopes of C7 are
6
also located within the NC2 and triple helical domain of C7. Based on the wide
autoantibodies reactivity of EBA and the characteristic of NC1 domain, we developed a
NC1 based enzyme-linked immunosorbent assay (ELISA) for EBA screening. Bullous
systemic lupus erythematosus (BSLE) has been reported to show autoantibodies against
C7 as well [14]. But with other musculoskeletal, cardio, hematological and renal
symptoms in BSLE, it is easy to be distinguished from DEB and EBA [31].
1.3 Skin Wound Healing and Fibrosis
Skin, a soft covering of vertebrates, is the largest organ in human. This multi-
layer composed integumentary system protects the underlying muscle, ligaments, fat,
bone and internal organs. From the exterior to internal, the layers are the epidermis,
dermis, and hypodermis and they are bound together by various structures to mainly
protect animals from pathogens and dehydration, regulate body temperature, and provide
sensations. Epidermis and dermis, the primary layers of the skin, are tightly connected
by BMZ, which is composed of a thin layer of fibers. Once the skin experiences insults
that are about to destruct its integrity or affect its function, it would try to restore its
original structure and function.
According to the time frame, skin wounds are usually classified as acute and
chronic wounds; however, no specific time frames have been clarified. Most chronic
wounds are derived from acute wounds that have slow or preventive wound healing. The
risk factors that prevent patient acute wounds from healing are various. A few examples
are peripheral artery disease, diabetes and chronic venous insufficiency.
From a macroscopic view, chronic cutaneous wounds affect 6.5 million patients
in the United States alone, and costing taxpayers $25 billion annually. Moreover, as a
consequence of skin wound healing, an additional $12 billion is spent annually treating
skin scarring [32, 33]. Wound healing is a complicated and highly dynamic process,
involving a great variety of cells and soluble mediators. Based on what we now know
about skin wound healing, it has five overlapping phases: (i) inflammation, (ii)
epithelialization, (iii) formation of granulation tissue, (iv) neovascularization, and (v)
wound contraction and extracellular matrix reorganization. From a microscopic view,
7
aside from the economic and time burden each patient who suffers due to the chronic
wound healing process, patients also experience pain and swelling from inflammations,
or from surgeries to either help wound closure, such as skin transplantation, or prevent
patients from death, such as severe ulcers causing amputation surgery as often seen in
diabetic patients [34].
The persistent healing in RDEB patients causes serious complications and low
quality of life. Once the skin encounters pressure or friction forces, RDEB patients
develop open or superficial bullous wounds [28], leading to inflammation. The
inflammation is either caused by broken blood vessels releasing blood with a pool of
platelets, or injured tissue secreting vasoactive and chemotactic factors, recruiting
inflammatory leukocytes to the injure site. Meanwhile, neutrophils and macrophages
clean off the bacteria and foreign particles from the wounds [35]. On a clean wound bed,
macrophages and monocyte release various growth factors to initiate the formation of
granulation tissue, including tumor necrosis factor alpha, colony-stimulating factor 1,
transforming growth factor β (TGF β), platelet-derived growth factor (PDGF) and more.
These growth factors are necessary and pivotal to the initiation and propagation of new
tissues[36].
Within a couple of hours after injury, re-epithelialization is initiated [37]. The
migrating epidermal cells secrete collagenase and plasminogen to degrade the ECM
proteins and collagens which could slow down or stop the epidermal cells from migrating
[38, 39]. 48 hours after the injury, new epidermal cells have been generated when both
inflammation and re-epithelialization are undergoing [34]. Besides the growth factors
released by epidermal cells, BMZ proteins are also generated in a tight order, with C7
being generated around two weeks [40, 41]. The inflammation and re-epithelialization
processes could take from a couple of days to and up to two to three months, depending
on the size and type of wounds.
The next step is a longer process, tissue remodeling, which starts four to five days
after the injury. The tissue remodeling process could take up to years and is divided into
a few different stages, formation of granulation tissue, neovascularization, and the wound
8
contraction and underneath ECM reorganization [34]. The granulation tissue is newly
formed stroma, which includes cells and temporary blood vessels. In detail, macrophages
keep secreting the growth factors necessary for angiogenesis and fibroplasia; fibroblasts
generate collagens to supply the new ECM; blood vessels transport the necessary oxygen
and nutrients. There are many biological agents involved in this comprehensive process.
However, the most significant growth factors during the formation of granulation tissue
are PDGF and TGF β. Currently, only the topical application of PDGF has been
approved by the Food and Drug Administration (FDA) to enhance skin wound healing.
Topical PDGF has been on the market for years, but beneficiaries are only limited to
diabetic patients, and proven to have only moderated efficacy. The provisional ECM is
composed by fibroblasts secreting fibrin, fibronectin, collagens, and hyaluronic acid [42].
Eventually a mature and stable collagenous ECM replaces the provisional ECM. During
neovascularization, the endothelial cell is the main cell line in the neovascularization. It
forms new blood vessels under the stimulation of self-secreted endothelial growth factors,
basic fibroblast growth factor secreted by fibroblasts, and acid. Once the granulation
tissue is formed, angiogenesis stops and the new blood vessels undergo apoptosis.
Wound contraction, the transformation from granulation tissue to mature
collagenous ECM, starts a week after wounding. Reorganization of the wound bed can
take up to a few years to finish, involving cells, ECM, and cytokines. Secreted TGF-β1,
TGF-β2 and PDGF stimulate part of regular fibroblasts differentiation into
myofibroblasts, which is responsible for wound contraction [43]. Both fibroblasts and
myofibroblasts could bind collagen matrix through intergrins and cross-link with collagen
bundles. Collagens are continually being synthesized and a relatively low rate of
degradations is expected in normal wound healing. The strength of wounds depends on
the amount of collagen bundles, which need a long time to synthesize and reorganize.
The rate of tensile strength is a parameter to determine the maturation of healed wound.
However, a healed wound skin would never be as strong as normal skin [44].
Skin fibrosis, the end stage of pathological wound healing, is caused by chronic
inflammation. This persistent immune response occurs in the whole process of skin
wound healing, sustaining the production of growth factors, proteolytic enzymes,
9
angiogenic factors and fibrogenic cytokines. All of these factors could stimulate the extra
deposition of connective tissue elements. Combinatorial signaling pathways are involved
in the wound healing process. They function either synergistically or counteractively to
maintain normal wound healing. However, the deregulation of different cytokines,
growth factors, and transcriptional regulators leads to imbalanced synthesis of ECM
proteins, ECM proteases and their inhibitors that cause fibrosis [45].
TGF β is a multifunctional cytokines and is synthesized by a number of cells
including monocytes, macrophages, and fibroblasts. It is also being called TGF β
subfamily, one of four subfamilies in the TGF β superfamily, since it includes three
isoforms, TGF β1, TGF β2, and TGF β3. All three isoforms bind with TGF β receptor I
(TGF β R-I) and TGF β receptor II (TGF β R-II), in order to start signal transduction, and
through either canonical Smad pathway or non-canonical pathways activate the
downstream gene transcriptions [46, 47]. TGF β pathway is one of the major growth
factor pathways and has been intensive studied. It is involved in cell growth, cell
differentiation, apoptosis, cellular homeostasis, and other functions in both embryo and
adult organisms [48]. TGF β is highly responsible for an abnormal outcome of wound
healing and excessive scars in initiation and progression of fibrogenesis. It also induces
the migration of fibroblasts, and the differentiation of fibroblasts to myofibroblasts [49].
Low amount of TGF β1 has been observed in fetal wounds that heal with less scar. [34].
Also tissue fibrosis has been reported which primarily attributed by TGF β1 isoform.
Moreover, the results of several in vivo animal fibrosis model studies demonstrated the
significance of Smads, especially Smad 3, in organ fibrogenesis [50, 51].
The healed wounds in RDEB patients are considered hypertrophic scars, which
appear in an abnormal and raised nature. Unlike the keloid scars, hypertrophic scars are
limited in the boundary of the original wound area. Researchers have found an up-
regulated TGF-β1 mRNA and a prolonged expression of TGF β receptors in the
fibroblasts isolated from hypertrophic scars [52]. Additionally, more studies suggested
that the receptor regulated Smad signaling, phosphorylated Smad 2 and Smad 3 signaling
are up-regulated in the hypertrophic scars. Hypertrophic scars are often seen in burn
patients, and researchers have also found that up-regulated TGF β in serum could be an
10
indicator for burn patients. The findings above suggest that TGF β pathways are highly
related to scar formation, especially in hypertrophic scars [53].
11
References:
1. Bentz, H., et al., Isolation and partial characterization of a new human collagen
with an extended triple-helical structural domain. Proc Natl Acad Sci U S A,
1983. 80(11): p. 3168-72.
2. Parente, M.G., et al., Human type VII collagen: cDNA cloning and chromosomal
mapping of the gene. Proc Natl Acad Sci U S A, 1991. 88(16): p. 6931-5.
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39. Bugge, T.H., et al., Loss of fibrinogen rescues mice from the pleiotropic effects of
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40. Larjava, H., et al., Expression of integrins and basement membrane components
by wound keratinocytes. J Clin Invest, 1993. 92(3): p. 1425-1435.
41. Clark, R.A., et al., Fibronectin and fibrin provide a provisional matrix for
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42. Greiling, D. and R.A. Clark, Fibronectin provides a conduit for fibroblast
transmigration from collagenous stroma into fibrin clot provisional matrix.
43. Welch, M.P., G.F. Odland, and R.A. Clark, Temporal relationships of F-actin
bundle formation, collagen and fibronectin matrix assembly, and fibronectin
receptor expression to wound contraction.
44. Levenson, S.M., et al., THE HEALING OF RAT SKIN WOUNDS.
45. Ghosh, A.K., S.E. Quaggin, and D.E. Vaughan, Molecular basis of organ
fibrosis: potential therapeutic approaches. Exp Biol Med (Maywood), 2013.
238(5): p. 461-81.
46. Sullivan, K.M., et al., A model of scarless human fetal wound repair is deficient in
transforming growth factor beta.
47. Clark, D.A. and R. Coker, Transforming growth factor-beta (TGF-beta). Int J
Biochem Cell Biol, 1998. 30(3): p. 293-8.
48. Pierce, G.F. and T.A. Mustoe, Pharmacologic enhancement of wound healing.
49. Wynn, T.A., Cellular and molecular mechanisms of fibrosis. J Pathol, 2008.
214(2): p. 199-210.
50. Zhao, J., et al., Smad3 deficiency attenuates bleomycin-induced pulmonary
fibrosis in mice. Am J Physiol Lung Cell Mol Physiol, 2002. 282(3): p. 585-593.
51. Lakos, G., et al., Targeted disruption of TGF-beta/Smad3 signaling modulates
skin fibrosis in a mouse model of scleroderma. Am J Pathol, 2004. 165(1): p. 203-
217.
52. Zhang, K., et al., Increased types I and III collagen and transforming growth
factor-beta 1 mRNA and protein in hypertrophic burn scar.
53. Tredget, E.E., et al., Hypertrophic scars, keloids, and contractures. The cellular
and molecular basis for therapy.
14
Figure:
Figure 1.1 The domain organization of a C7 alpha chain and how C7 molecules
assemble into AFs. A C7 molecule is composed of three identical α chains stabilized by
interchain disulfide bonds. Each α chain consists of an amino-terminal, NC1 domain and
a carboxyl-terminal NC2 domain. A triple helical collagenous domain connects these
two domains. In addition, there is a hinge region, TH in the middle of the triple helical
domain, which can be digested by pepsin. Several sub-modules in NC1 domain that are
homologous to some adhesive proteins, including CMP on the amino-terminus, next is
nine repeats of FN-III domains, and VWFA on the carboxyl terminus. 1&2. NC2 domain
with a short adjacent triple helical sub-domain is responsible for initiating triple helix
assembly and antiparallel dimer formation. 3. Within the extracellular space, the C7
molecules form antiparallel, tail-to-tail dimers, and the overlapped NC2 parts are
removed during the proteolytic process. 4. Then the dimers aggregate to form a structure
called AFs providing the stability of the epidermal and dermal adhesion.
15
Chapter II
Intradermal Injection of Recombinant Human Type VII Collagen Corrects the
Disease Phenotype in a Murine Model of Dystrophic Epidermolysis Bullosa
2.1 Summary
This chapter discusses the feasibility of C7-based protein therapy for DEB using
preclinical animal model, a C7 knock out mouse model. DEB is a family of inherited
mechano-bullous disorders caused by mutations in the human C7 gene. These mutations
cause perturbations in AFs, structures that hold the outer epidermal layer of skin onto the
inner dermal connective tissue layer. Without sufficient functional AFs, the epidermis
separates from the dermis. Children with DEB are born with skin fragility and
continuous blistering of the skin in trauma-prone sites. Eventually, most of these
children die from aggressive squamous cell carcinoma at the sites of chronic blistering
and scarring. The C7 null DEB mouse recapitulates the clinical, genetic and
ultrastructural features of human RDEB. We have demonstrated the feasibility of ex vivo
gene therapy strategies for DEB using a lentiviral-based gene transfer approach, and also
shown that the intradermal injection of gene-corrected DEB fibroblasts (cell therapy),
recombinant human C7 (protein therapy), or lentiviral vectors expressing human C7
(vector therapy) into a DEB human skin equivalent engrafted onto a mouse achieves
long-term expression of C7 that incorporates into the skin’s BMZ and corrects the DEB
phenotype. Because all of these studies were conducted in immunodeficient mice, they
did not address the potential immune responses induced by the introduction of viral
vectors, the transgene, or the recombinant protein. Prior to testing these approaches in
DEB patients, we wished to validate them in a preclinical animal model to determine the
safety, efficacy, and potential immune responses.
In this chapter, we evaluated the feasibility of C7 protein therapy with intradermal
injections into C7 null mice. After intradermally injecting human rC7 into new born
DEB mice with severe skin blistering and fragility, we found that human rC7 migrated
and incorporated into the mouse’ BMZ and formed AFs. Accordingly, the DEB mice
phenotypes were significantly improved with decreased skin blistering, increased
adherence of dermal epidermal junction, and the most pivotal benefit, the prolonged life
16
expectancy. Interestingly, although anti-human C7 antibody is induced after rC7
injections, however, no adverse effects were observed in the animals. Lastly, we showed
that anti-C7 antibody production was significantly prevented when the mice were treated
with an anti-CD40L monoclonal antibody, the major histocompatibility complex related
protein-1 (MR1). Our studies provide the first evidence for using protein therapy to
correct a skin disease in a preclinical animal model. We conclude that protein therapy
may be feasible for the treatment of human patients with DEB.
2.2 Results
2.2.1 Restoration of C7 in the DEB mouse’s BMZ
A C7 knockout murine model, which recapitulates the genetic, ultra-structural,
clinical characteristics of the most severe human RDEB, was utilized to evaluate the
feasibility of C7 protein therapy for DEB [44]. As shown in Figure 2.1a and 2.1b, the
C7 null mice displayed hemorrhagic blisters on the paws, neck and ventral area.
Hematoxylin & Eosin (H&E) stain results in Figure 2.1c showed dermal-epidermal
separation and immunostaining with anti-C7 antibody revealed an absolute absence of C7
at the BMZ. On the other hand, the skin from wild type littermate demonstrated strong
C7 staining at the BMZ. Without treatments, the DEB mice died from complications
caused by extensive blistering within a week.
To evaluate whether protein therapy could reverse DEB mouse phenotypes, from
the day of their birth, we intradermally injected purified human rC7 into their dorsal area.
Skin biopsies were obtained one week after the first injection from the injection site and
subjected to immunostaining with a polyclonal rabbit antibody that recognized the amino
terminal non-collagenous domain NC1 of C7. We observed that injected rC7 transported
and incorporated into the BMZ shown in Figure 2.2a. The C7 expression was strongest
near the injection site, and gradually faded away on the surrounding areas. In some
severely affected animals, we not only detected the C7 expression on the injected area but
also in remote areas far from injection sites such as the neck, abdomen, and paws. This
data indicated that the poor dermal epidermal adherence of skin in severe affected DEB
17
mice allowed injected rC7 to transport into other areas distally. Additionally, as shown in
Figure 2b, injected rC7 was detected as early as 6 hours after one injection and
maintained for at least 7 months with continued injections weekly.
To determine the half-life of injected rC7 in vivo, we only conducted multiple
injections into DEB mice (n=10) in the first week after birth and stopped injections
afterward. In skin samples obtained at various time points, we still were able to detect
the injected rC7 up to two months following the last treatment. At 14 weeks after the
final injection, the biopsies no longer showed presence of rC7 in the skin of the animals.
These data suggested that rC7 has a half-life for at least two months or longer in vivo.
2.2.2 Absence of C7 in the blood and other organs of DEB mice after protein therapy
We observed that rC7 could distribute distally from the injection sites, and
therefore it is possible that injected rC7 was transported through the animals’ circulation
system. To test this hypothesis, we performed immunoblot analysis on sera obtained
from DEB mice with various treatment periods. As shown in Figure 2.3a, there is no
rC7 detectable rC7 found in the sera. Moreover, to decide whether intradermal injected
C7 transported to tissues other than skin, we performed immunofluorescence staining to a
series of internal organs from rC7 injected mice. As exhibited in Figure 2.3b, there were
no rC7 detected in the heart, liver, lung, brain, kidney, spleen, and small intestine.
2.2.3 Formation of AFs by injected human rC7
Reduced, malfunctioned or absent AFs is the reason for DEB patients presented
with fragile skin. Reversing the major clinical symptom of DEB is a crucial objective of
all DEB targeted therapies. We performed multiple injections of DEB mice with 60 μg
of rC7, biopsied skin from injected site and subjected skin sections to immuno-electron
microscopy with an anti-NC1 antibody. As shown in Figure 2.4, the injected human rC7
incorporated into the mouse’s BMZ, oriented correctly and formed AF structures,
therefore verifying the correction of the major clinical symptom in the ultra-structural
level.
2.2.4 Increased survival of DEB mice by protein therapy
18
With the complications of tremendous blistering, DEB mice would not survive for
more than a week without treatment. For that reason, we also determined the survival
rate as an index to evaluate the efficacy of rC7 intradermal injections. The Kaplan-Meier
curve in Figure 2.5a showed that rC7 injected mice had significantly increased life span,
and the median survival was 5 weeks for 45 rC7 treated mice (range: 1-28 weeks), while
comparing to 1 week (range: 0-1 week) for 30 untreated DEB mice (P<0.001, long-rank
test). Notable is that 7 of the rC7 injected mice survived over 12weeks among the 45
treated mice. The mouse showing in the Figure 2.5b has survived 22 weeks, and has
been administrated multiple injections with cumulative 250 μg of rC7.
Immunofluorescence staining with anti-NC1 antibody revealed displayed the strong C7
staining at the BMZ of the skin biopsies, and H&E staining of C7 injected skin showed
the good dermal-epidermal adherence, both of the results showed similarity to the normal
mouse skin.
To exclude the possibilities that the survival of DEB mice can be extended by any
protein, and also to confirm the specificity of rC7 injections, we injected equal dose as
rC7 of bovine serum albumin (BSA) into fifteen DEB mice. Fourteen out of fifteen BSA
injected mice died within 5 days after birth, and one of the fifteen died at day 10. Hence,
we confirmed that the increased survival rate of rC7 injected DEB mice are specifically
due to the treatment of rC7. In summary, we injected a total of 45 DEB mice with rC7 at
cumulative dose range between 20 μg to 300 μg, 88% of them had rC7 restored at the
BMZ of the dorsal skin, and exhibited a significant increased life span in general.
2.2.5 Production of anti-C7 antibodies after injection of human rC7
Immune responses would be provoked when a new or foreign protein is
introduced into the animals that do not express this protein and see it as foreign. In this
case, we expected that the unwanted immune responses such as production of anti-C7
antibody would increase in rC7 treated DEB mice. To examine whether the human rC7
treated DEB mouse has produced anti-C7 antibodies, we collected the sera of six human
rC7 injected DEB mouse at various time points, and subjected them to NC1 based ELISA
to detect the presence of anti-C7 antibodies [45]. All sera from six human rC7 injected
19
mice have produced anti-C7 antibodies within the first 30 days after injection and the
titers occurred in a steadily increasing fashion exhibited in Figure 2.6a. We also
performed indirect immunofluorescence staining with sera either on mouse or salt-split
human skin to determine the titers. As shown in Figure 2.6b, sera from human rC7
injected DEB mice showed mouse immunoglobulin G (IgG) antibodies staining on both
skin substrates, to be particular, the DEJ of normal mouse skin and the dermal side of the
salt-split human skin.
Immunologically, the presence of IgG antibodies targeting C7 would induce a
chronic autoimmune sub-epidermal blistering disease, named epidermolysis bullosa
acquisita (EBA), which exhibits blistering and scars on skin. To determine whether the
induced anti-C7 antibody bind to skin, we biopsied skin from injection sites at various
time points after injections and subjected them to direct immunofluorescence staining
with antibodies to murine IgG and C3 complement. As shown in Figure 2.7a, there is no
murine IgG or C3 complement detected in spite of that, and we observed abundant
human rC7 presence at the BMZ as revealed by immunostaining with NC1 antibody on
the same samples. A small population of EBA patients has been reported to exhibit
immunoglobulin A (IgA) on patients’ skin. We tried and did not detect other
immunoglobulin isotypes such as IgA or IgM in the BMZ of rC7 injected mouse skin
Although induced anti-C7 antibodies in rC7 injected DEB mice did not bind
directly to the protein at the BMZ, it is possible that the induced anti-C7 antibodies
circulating in the blood stream could bind to the newly injected human rC7 and prevent
its incorporation into the BMZ, thus diminishing the therapeutic efficacy of future rC7
injection. To determine that, we selected four DEB mice, which contained anti-C7
antibodies in sera as detected by ELISA, and then injected 20μg human rC7 on their
ventral skin. Ventral skin is distant away from original injections area where we never
had observed any human rC7 deposition. After one week, we biopsied the ventral area
and subjected it to immunofluorescence staining. Interestingly, the newly injected human
rC7 was still able to incorporate into the BMZ of ventral skin, as shown in Figure 2.7b.
In contrast, C7 expression was not detected on ventral sides of DEB mice, which did not
receive rC7 injection. These data showed that the presence of anti-C7 antibodies in the
20
circulation do not prevent new supplement of human rC7 incorporation into the BMZ of
skin. In conclusion, the induced anti-C7 antibodies may not be pathogenic and unlikely
to provoke EBA in the treated mice, despite the presence of circulating anti-C7 antibodies
in their blood streams [46, 47].
2.2.6 Inhibition of anti-C7 antibody formation by MR1
Although the anti-C7 antibody may not be pathologic to the animals, we still
attempted to find an immuno-suppressant that can inhibit the induced anti-C7 antibodies.
We utilized MR1, the anti-CD40L monoclonal antibody. In immunology research, MR1
has been widely used to block the CD40-CD40L interaction to abrogate autoimmune
diseases or to induce the transplantation tolerance [48]. Three DEB mice in each group
were treated either with MR1 or control IgG intraperitoneally at days 0, 2, 4, 7, 14, 21
and 28 prior to human rC7 injection. We collected serum samples at day 30, 60 and 90,
and subjected them to NC1 based ELISA as described [45]. As shown in Figure 2.8, the
MR1 treated DEB mice did not have detectable anti-C7 antibodies at any of the time
points, however, the control IgG treated DEB mice produced high levels of anti-C7
antibodies at day 30 and was maintained after that. The obtained data indicate that MR1
is effective in preventing the production of murine anti-C7 antibodies, which is induced
by intradermal injection of human rC7 to COL7A1 null mice.
2.3 Discussion
In this study, a C7 knockout mouse model (COL7A1-/-) was used to evaluate the
feasibility of intradermal injection of human rC7 for protein therapy of DEB. The basis
for this study is that Chen’s lab has the ability to purify human rC7 in large amounts from
gene-corrected RDEB fibroblasts [49]. Our study revealed that intradermal injection of
human rC7 stably incorporated into the BMZ of the DEB mouse skin, and formed AFs.
As a consequence, the DEB phenotype has been corrected, namely improving the
adherence of the BMZ of DEB skin, preventing blistering on treated mice, and
significantly prolonging the life span of DEB mice. This was the first evidence ever to
show that protein therapy can correct a genetic skin disease in a preclinical animal model.
Due to an 85% identity and 90.4% homology at the amino acid level between mouse and
21
human C7, our studies show that human rC7 could bear the responsibility for its murine
counterpart [15]. Previously we conducted human rC7 intradermal injection experiments
on intact normal mouse skin or human RDEB skin equivalent transplantation model and
showed that the rC7 incorporated into the BMZ and corrected the RDEB phenotypes
[49]. Nevertheless, we only observed localized staining close to the injection areas, and
did not observe C7 staining in the surrounding tissues in our previous studies. In
contrast, in the present study with C7 null DEB mice, we noticed that 25% of the human
rC7 injected DEB mice were not only showing C7 staining in the injected area, but also
had detectable C7 at vertical distant areas, such as paws and necks. Our theory to this
phenomenon is that the natural jeopardized dis-adherence of BMZ on DEB mouse skin
provided an “outlet” to allow the human rC7 to seep through the entire body, making the
localized intradermal administration become a “systematical” treatment to DEB mouse
model. It also well explained why we detected the C7 expression from a distance more
often in the mice with the most severe clinical phenotype at birth.
Skin is the largest organ in mammals; its major functions are protecting internal
organs from pathogens, preventing excessive water loss, insulation and temperature
regulation [50]. With fragile and traumatized skin, DEB mice usually die within a week
after birth due to infection or/and water loss or/and low body temperature. According to
reports, fluid in blisters of some pups reach up to 20% of their average body weight [44].
The critical skin conditions making the first week is vital to the new born DEB mice;
however, once they pull through the critical stage with the help of intradermal supplied
rC7, the animals began to show reduced blistering, and over 88% of treated DEB mice
exhibited a more intact skin with significant prolonged life span.
Up until the 8
th
week, rC7 treated DEB mice were showing similar levels of
growth and activeness while compared to their wild type littermates. However, the rC7
treated DEB started revealing retarded growth after 8 weeks. This occurred due to the
intradermal injection treatment only being able to treat skin blistering, and not reaching
into the oral mucosa to relieve the clinical symptoms. Moreover, mice need to take solid
food once they have been weaned off from their mothers around 6-8 weeks. Since
intradermal injected rC7 did not reach the oral mucosa, the presence of blisters and
22
erosions at the oral mucosa of the rC7 treated mice prevented them for eating solid food
and resulted in growth retardation and eventual of death from the malnutrition.
This study has a potential side effect of induced autoimmunity in the experimental
animals. Preventing immune responses against the exogenous agents is an important
issue of all protein replacement therapy with a newly introduced therapeutic agent.
Especially for the recessive inherited diseases where the expression of newly introduced
protein is completely missing, preventing immune responses becomes an unavoidable
critical topic. Because the animals’ immune system has never been exposed to the
missing protein, newly introduced protein would be partially or completely recognized as
a foreign protein and provoke the unnecessary immune responses. To DEB in particular,
the importance of preventing anti-C7 antibody production is further emphasized by the
fact that autoantibodies to C7 are related to EBA [46, 47]. The autoantibodies bind to C7
within AFs and cause the decrease of AF in the patients’ skin. It is possible that
intradermal injected human rC7 corrected the DEB phenotype and cured the disease in
patients, while introduced patients to other immune diseases with similar symptoms. In
this chapter anti-C7 antibodies were detected in the bloodstream of DEB mice
intradermally injected with human rC7 at one month after initial injection and the titers of
antibodies increased steadily over the 6 months period. Nevertheless, the anti-C7
antibodies did not deposit onto BMZ of skin, although the abundant human rC7 are over
there [51]. These data indicate that anti-C7 antibodies may not be pathogenic. In
addition, we demonstrated that the presence of circulating anti-C7 antibodies did not
prevent newly supplied human rC7 from incorporating onto the BMZ of the DEB mouse
skin.
In our previous EBA studies, we passively transferred human or rabbit antibodies
against C7 into immune-competent mice and induced an EBA-like bullous disease in the
mice, demonstrating the pathogenicity of anti-C7 antibodies in the induction of EBA. It
is interesting to note that anti-C7 antibodies detected in human rC7 injected mice were
not pathogenic and did not induce EBA. After analyzing the titers of anti-C7 antibodies
by indirect immunofluorescence staining performed on normal human or salt split human
skin, we found that the antibody titers in the sera of the rC7 injected DEB mice are at
23
1:20-100. They are significantly lower than 1:10,000-20,000 as seen in our previous
passive transfer studies [52, 53]. This could explain why there is no detectable mouse
anti-C7 IgG deposited in the skin of these mice when examined by direct
immunofluorescence.
The level of the host immune responses to the protein therapy depends on the
immunogenicity of the protein and the gene mutation of the host. For RDEB patients, the
patients who do not have any C7 expression are expected to have the most intense
immune response, and the rest of RDEB patients’ who have reduced or mutated C7
should experience less intense responses. Therefore, we are expecting more intense
immune response from RDEB, generalized, severe patients, and the least immune
response from the patient who retained the NC1 domain of C7. Based on previous
studies, more than 60% of RDEB patients have expressed NC1 domain [24].
CD40 is mainly expressed on antigen presenting cells (APC), and the CD40-L is
primarily expressed on activated T cells. The interaction of CD40 and CD40-L could
stimulate T-cell activation, B-cell proliferation and immunoglobulin secretion [48, 54,
55]. The monoclonal antibody of CD40-L, MR1 is widely utilized in transplantation
surgery to induce tolerance and delivery to autoimmune disease patients to abrogate
them. We used MR1 to inhibit murine anti-C7 antibody stimulated by human rC7
injection in RDEB murine model. Furthermore, no adverse effect has been observed with
MR1 suppression in mice. Besides the usage of MR1 is simply related to CD40
molecule, which is primarily responded to activated T cells, the understanding of auto-
antigen structure is not necessary [56]. Our study indicated that utilizing of MR1, the
CD40-L antibody was effective to prevent autoantibodies production in human rC7
treated RDEB murine model, and thus might be helpful in the clinical trial of intradermal
injection of C7 protein into RDEB patients in the near future.
Proteinaceous agents have limited half-lives in vivo in general, so the related
therapies require frequent administration to maintain the therapeutic efficacy. Treatments
for hemophilia and arthritis are the best examples to explain this dilemma. In previous
studies we found that injected rC7 stably incorporated into the BMZ of human RDEB
24
skin equivalent and sustained for up to 3 months after a single injection. Moreover, in
this study we had consistent results that injected human rC7 stably incorporated into the
BMZ of DEB mouse and persisted for at least two months [57, 58]. By summarizing the
findings, we believe that human rC7 has a long half live in vivo and is an ideal
therapeutic agent for its efficacy, stability and safety. In this case, we could inject
amounts of rC7 that form abundant AFs, which could persist for decent time.
In summary, our studies provide evidence that intradermally injected human rC7
could incorporate into the BMZ of DEB mouse skin, form AFs, improve the poor
adherence of dermal epidermal junction of DEB skin, and significantly prolong the life
span for the DEB mouse from one week to an average of five weeks. Among all the
therapies for RDEB, intradermal delivery of C7 protein is (i) technical feasibility, human
rC7 is relatively easy to purify in large quantities, (ii) conceptual straightforward,
intradermal injection of C1 has been applied in clinics for years for rhytide effacement,
(iii) therapeutically effective and (vi) showed manageable immune responses. This study
has laid a strong foundation for clinical trial of testing the therapeutic efficacy and safety
of intradermal injection rC7 into the RDEB patients.
2.4 Materials and Methods
Animal studies.
COL7A1+/– animals were developed as described previously and maintained at
the animal facilities of the University of Southern California, Los Angeles, under
guidelines for the care and use of animals in research [44]. All animal studies were
conducted using protocols approved by the University of Southern California Institutional
Animal Use Committee. The genotype of the Col7a1+/– animals was verified by PCR of
the C7 gene with a template of genomic DNA from tail samples. Col7a1+/– animals
were clinically normal and indistinguishable from the wild type littermates (Col7a1+/+).
Heterozygous mice were intercrossed to produce Col7a1–/– null (Col7a1) offspring.
DEB mice were readily identified at birth by the large fluid-filled blisters developed
primarily on the ventral side of the animals and the large hemorrhagic blisters on their
paws. Their genotype was further confirmed by PCR. For protein therapy, we
25
intradermally injected 10μg of purified recombinant human C7 suspended in 30ul of
phosphate-buffered saline into the dorsal back skin of DEB mice (n = 45) once every day
for the first week and then weekly thereafter using a 28 1/2 gauge needle. Therefore,
depending on how many days each of these DEB mice survived (range from 4 days to 7
months), the cumulative total amounts of C7 injected into each DEB mouse varied from
20 to 300μg. The injected animals were photographed daily and assessed for therapeutic
efficacy by monitoring weight gain, survival, and reduced skin blistering. At various
times after injections, mouse skin biopsies were obtained from the whole body (including
injected and uninjected areas) and subjected to immunostaining using a rabbit polyclonal
antibody recognizing NC1 domain of human C7, as described in the following text.
Histological sections of mouse’s skin were fixed in 10% buffered formalin and stained
with hematoxylin and eosin.
Blood samples were taken at the indicated times from the retro-orbital plexus
using a sterile 200ul filter tip and then stored at 4 °C overnight.
Immunofluorescence staining and ultrastructural analysis of tissue.
Five-micrometer thick sections of the OCT-embedded tissues were cut on a
cryostat; applied cold acetones and air-dry 5min for fixation. Sections of skin from mice
intradermally injected with C7 were incubated with a rabbit polyclonal antibody
recognizing both mouse and human C7, followed by a Cy3-conjugated goat anti-rabbit
IgG (Sigma, St. Louis, MO). Working dilutions were 1:500 for the primary antibody and
1:200 for the secondary antibody. Immunolabeling of the tissue was performed using
standard immunofluorescence methods as described previously. Slides were mounted
with 40% glycerol. Pictures from stained sections were taken using a Zeiss Axioplan
fluorescence microscope equipped with a Zeiss Axiocam MRM digital camera system.
Immunogold electron microscopy was performed on the mouse skin using a
standardized method as described previously [59]. To assess human anchoring fibril
formation and ultrastructure, 40 um sections were fixed in 0.1% glutaraldehyde, rinsed in
0.15 mol/l Tris, pH 7.5, and then incubated in our polyclonal anti-NC1 antibody followed
by 5nm gold secondary antibody and enhancement as described [60, 61].
26
ELISA using recombinant NC1.
The production of circulating anti-C7 antibodies was evaluated by ELISA, using
the recombinant NC1 domain of C7, as previously described [45]. Briefly, 96-well
microtiter plates (Immulon-4; Dynatch Laboratory, Alexandria, VA) were coated with
purified recombinant NC1 at a concentration of 1.5 μg/ml (0.15 μg/well) in 20 mmol/l
carbonate buffer, pH 9.3, overnight at 4 °C. The plates were washed three times with 20
mmol/l phosphate, pH 7.4, 150 mmol/l NaCl containing 0.05% Tween-20 (PBST).
Nonspecific binding was reduced by blocking the plates with PBST containing 1% BSA
at room temperature for 2 hours. Coated wells were subsequently incubated with sera
obtained from DEB mice (dilution at 1:100 in PBST with 1% BSA) at room temperature
for 2 hours. Wells were washed three times and incubated with alkaline phosphatase–
conjugated goat anti-mouse IgG (Cappel, Aurora, OH) diluted in PBST with 1% BSA (1:
1,000) for 1 hour. The plates were then washed with PBST three times, and p-
nitrophenylphosphate (Bio-Rad, Melville, NY) substrate was added and allowed to react
for 4-8 minutes. Optical density was measured by absorbance at 405 nm (Bio-Tek
Instruments, Winooski, VT). We calculated the mean and standard deviation for this
ELISA using optical density values of control sera from 12 normal mice. On the basis of
the mean, we set the cutoff values for definite positive reactivity as 0.2.
To evaluate whether there are any anti-C7 antibodies deposited directly in the
skin, DEB mouse skin tissues from both injected and un-injected areas were subjected to
direct immunofluorescence staining using fluorescein isothiocyanate–conjugated goat
anti-mouse IgG, IgM, or IgA (Sigma, St. Louis, MO) as previously described [46].
MR1 and control IgG treatment.
Hamster anti-mouse CD40L monoclonal antibody MR1 was purchased from
Taconic Farms (Taconic Farms. Germantown, NY). The DEB mice were injected
intraperitoneally with either MR1 (n = 3) or control hamster IgG (n = 3) (Cappel Product,
Aurora, OH). The dose of MR1 was determined based on previous reports as well as our
dose–response study [56, 62, 63]. We used 20μg/g body weight at day 0 and 10μg/g
body weight per mouse at days 2, 4, 7, 14, 21, and 28. Serum samples from these mice
27
were collected once a month and analyzed with ELISA using human NC1, as described
earlier[45].
28
References:
1. Heinonen, S., et al., Targeted inactivation of the type VII collagen gene (Col7a1)
in mice results in severe blistering phenotype: a model for recessive dystrophic
epidermolysis bullosa. J Cell Sci, 1999. 112 ( Pt 21): p. 3641-8.
2. Chen, M., et al., Development of an ELISA for rapid detection of anti-type VII
collagen autoantibodies in epidermolysis bullosa acquisita.
3. Woodley, D.T., et al., Identification of the skin basement-membrane autoantigen
in epidermolysis bullosa acquisita.
4. Woodley, D.T., et al., Epidermolysis bullosa acquisita antigen is the globular
carboxyl terminus of type VII procollagen.
5. Datta, S.K. and S.L. Kalled, CD40-CD40 ligand interaction in autoimmune
disease.
6. Woodley, D.T., et al., Injection of recombinant human type VII collagen restores
collagen function in dystrophic epidermolysis bullosa. Nat Med, 2004. 10(7): p.
693-695.
7. Kivirikko, S., et al., Cloning of mouse type VII collagen reveals evolutionary
conservation of functional protein domains and genomic organization. J Invest
Dermatol, 1996. 106(6): p. 1300-6.
8. Madison, K.C., Barrier function of the skin: "la raison d'etre" of the epidermis. J
Invest Dermatol, 2003. 121(2): p. 231-41.
9. Woodley, D.a.C., M, fitzpatrick's dermatology in general medicine. 2007:
McGraw-Hill Companies.
10. Woodley, D.T., et al., Evidence that anti-type VII collagen antibodies are
pathogenic and responsible for the clinical, histological, and immunological
features of epidermolysis bullosa acquisita.
11. Woodley, D.T., et al., Induction of epidermolysis bullosa acquisita in mice by
passive transfer of autoantibodies from patients.
12. Ortiz-Urda, S., et al., Type VII collagen is required for Ras-driven human
epidermal tumorigenesis. Science, 2005. 307(5716): p. 1773-1776.
13. Stein, C.S., I. Martins, and B.L. Davidson, Long-term reversal of
hypercholesterolemia in low density lipoprotein receptor (LDLR)-deficient mice
by adenovirus-mediated LDLR gene transfer combined with CD154 blockade.
14. Ranheim, E.A. and T.J. Kipps, Activated T cells induce expression of B7/BB1 on
normal or leukemic B cells through a CD40-dependent signal.
15. Ohyama, M., et al., Suppression of the immune response against exogenous
desmoglein 3 in desmoglein 3 knockout mice: an implication for gene therapy.
16. Burgeson, R.E., Type VII collagen, anchoring fibrils, and epidermolysis bullosa. J
Invest Dermatol, 1993. 101(3): p. 252-255.
17. Pelkonen, R. and K.I. Kivirikko, Hydroxyprolinemia: an apparently harmless
familial metabolic disorder.
18. Keene, D.R., et al., Type VII collagen forms an extended network of anchoring
fibrils. J Cell Biol, 1987. 104(3): p. 611-21.
19. Sakai, L.Y., et al., Type VII collagen is a major structural component of
anchoring fibrils. J Cell Biol, 1986. 103(4): p. 1577-1586.
29
20. Sakai, L.Y. and D.R. Keene, Fibrillin: monomers and microfibrils. Methods
Enzymol, 1994. 245: p. 29-52.
21. Yang, Y., et al., Transient subversion of CD40 ligand function diminishes immune
responses to adenovirus vectors in mouse liver and lung tissues.
22. Durie, F.H., et al., Prevention of collagen-induced arthritis with an antibody to
gp39, the ligand for CD40.
30
Figures:
Figure 2.1 Clinical, histological, and immunological presentation of DEB mice.
DEB mice show characteristic clinical features of the disease including skin fragility,
hemorrhagic blisters, erosions, and large fluid-filled bullae primarily on the ventral side.
(A) Typical large hemorrhagic blisters are seen on paws and (B) large blisters are seen on
the ventral side. (C) Hematoxylin and eosin staining of skin from DEB mice revealed
separation of the epidermis (e) from the dermis (d). (D&E) Immunofluorescence staining
of the tissues obtained from DEB mouse skin (D) and its wild-type littermate’s skin (E)
with an affinity-purified polyclonal antibody recognizing the NC1 domain of C7. Note
the sub-epidermal separation and absence of C7 staining in DEB mice compared with the
strong C7 staining of the BMZ in normal mice.
31
Figure 2.2 Presence of C7 in DEB mice following protein injection. (a) DEB mice
were intradermally injected on their dorsal surface with 10μg rC7 once every day for 4
consecutive days, and tissue sections obtained from skin at various distances from the
injection site 1 week after injections were subjected to immunostaining using an anti-
NC1 antibody. Panels A, B, and C are biopsies taken from the immediate injected area,
near by and far away, respectively. Note that the injected human rC7 migrated from the
dermis and incorporated into the DEB mouse basement membrane zone at the injected
dorsal side. Interestingly, some biopsies obtained from sites remote from the injection
sites, such as neck (panel D), abdomen (panel E), and paw (panel F), also revealed C7
staining. (b) Dorsal side of skin from DEB mice injected intradermally with 10μg of
purified human rC7 once every day for the first week and then weekly thereafter was
stained with an anti-NC1 antibody. Panels A, B, and C are biopsies taken 6 hours, 15
weeks, or 28 weeks, respectively. For panel D, DEB mice (n = 10) were injected
with 60 μg rC7 for the first week only, and tissue sections obtained from the skin at 8
weeks after initial injections were subjected to immunostaining using an anti-NC1
antibody. e, epidermis; d, dermis.
Figure 2.3 Absence of C7 in blood stream and internal organs. (a) Sera were taken
from either control wild-type mice (WT) without injection or DEB mice that were
injected with rC7 once every day for the first week and then weekly thereafter at the time
indicated and subjected to 6% SDS-PAGE followed by immunoblot analysis using an
anti-NC1 antibody. 150ng of purified rC7 was run as a control (Con). The positions of
full-length 290-kd C7, 70-kd mouse IgG heavy chain (mIgGH), and molecular weight
markers are indicated. (b) Tissue distributions of intradermally injected C7. Four weeks
32
after injection of C7, necropsies were performed on the DEB mice (n = 6), and tissue
sections obtained from brain, kidney, liver, lung, spleen, heart, small intestine (SI), and
skin were subjected to immunostaining using an anti-NC1 antibody. Note that the
injected C7 was readily detected in the skin, but not in any other organs.
Figure 2.4 Immunoelectron microscopy of
mouse skin injected with human rC7.
Immunogold labeling of DEB mouse skin after
injections with 60μg of rC7 at 2 weeks after
injection was performed using an anti-NC1
antibody. Note that human C7 incorporated into
the mouse BMZ and formed AFs, ends of
which are decorated by gold particles
(arrowheads). D, dermis; E, epidermis; HD,
hemidesmosome. Scale bars = 400nm.
Figure 2.5 Improved survivals of DEB mice by C7 injections. (a) Kaplan–Meier
curves showing survival comparison between untreated (n = 30) and C7-treated (n = 45)
DEB mice. The curves indicate a significant increase in the survival of rC7-treated mice
in comparison with untreated DEB mice (P < 0.001). (b) Picture showing 1-day-old
DEB mouse with hemorrhagic blisters on paws (panel A)—the same mouse survived past
22 weeks after injections with cumulative 250 μg of rC7 (panel B). Immunofluorescence
staining with anti-NC1 antibody demonstrated strong C7 staining at the BMZ of the 22-
33
week-old treated mouse (panel C) and histological analysis revealed apparently normal
association of the epidermis and dermis at the cutaneous BMZ (panel D).
Figure 2.6 Anti-C7 IgG productions in DEB mice after protein therapy. (a) Anti-C7
IgG production was measured by NC1-based ELISA over time after intradermal
injections with human rC7. Anti-C7 IgG was detected at 30 days and increased steadily
thereafter in C7-treated mice (mean ± SD, n = 6). (b) Immunolabeling of mouse and
human skin with serum from mice injected with C7. Sections of normal mouse skin (A)
and salt-split human skin (B) were stained with mouse serum obtained from mice injected
with C7 at a dilution of 1:20. Note that circulating antibodies labeling the basement
membrane zone of mouse skin and the dermal floor of salt-split human skin were found
in the serum samples from C7-injected mice. OD, optical density.
34
Figure 2.7 Anti-C7 IgG neither bound to the skin directly nor inhibited further
BMZ incorporation of newly injected rC7. (a) Cryosections were obtained from the
back skin of RDEB mice treated with C7 for various times
as indicated and labeled with FITC conjugated goat anti-
mouse IgG (α-mIgG), goat anti-mouse C3 antibody (α-
mC3), or with an anti-NC1 antibody (α-NC1),
respectively. Please note that injected C7 was found at the
BMZ of DEB mice for all the time points (lower panels).
In contrast, no deposits of mouse- anti-C7 IgG (top
panels) or murine C3 (middle panels) were detected in the
BMZ of treated mice. (b) Incorporation of subsequently
injected rC7 into the mouse’s BMZ regardless of the
presence of C7 antibodies. DEB mice (n = 4) that had
been injected with C7 and were found by ELISA to have
anti-C7 IgG in their blood were re-injected with 10 μg of
C7 at remote sites where C7 was not expressed.
Immunofluorescence staining with an anti-NC1 antibody
was performed on mouse skin 1 week after injection at
both re-injected (panel A) and previously un-injected
abdominal areas (panel B). Please note that the newly
injected C7 still migrated from the dermis and incorporated into the mouse’s BMZ.
35
Figure 2.8 Suppression of
anti-C7 antibody
production by Mr1
treatment. All of the control
IgG-treated mice (n = 3)
developed anti-C7 IgG about
4 weeks after protein
injection, and this IgG was
maintained at high levels for
>3 months. This immune
reaction was effectively
suppressed when DEB mice were treated with MR1 (n = 3) at the time of protein
injection and continued at specific time points (arrows) for 30 days, as described under
Materials and Methods.
36
Chapter III
Topical Application of Recombinant Type VII Collagen Incorporates Into the
Dermal-Epidermal Junction and Promotes Wound Closure
3.1 Summary
RDEB patients consistently experience open wounds and erosions throughout
their lives caused by inherited skin fragility. It is unavoidable that healed wounds are
causing redundant scars as well. Those scars will cause many complications in RDEB
patients. Some examples of complications are, the fibrotic fusion of digits, joint
contractures of limbs, the conglutination of lips, fixed tongue, the stenosis and synechia
of esophagus, poor dentition and nutritional deficiencies. For patients aged between 20
and 30, the persistent chronic wound healing coupled with fibrosis in RDEB patients
have been considered as a major reason for developing aggressive squamous cell
carcinomas.
In this study, we investigated whether topically applied rC7 would be helpful in
enhancing wound healing. By using full thickness wound healing model in athymic nude
mice, we discovered that topically applied rC7 stably incorporated into the BMZ of
healing wounds. Moreover, during the wound healing process, the rC7 treated wounds
exhibited accelerated wound closure and less scar formation. Topical rC7 decreased the
expression of pro-fibrogenic TGF-β2 and increased the expression of anti-fibrogenic
TGF-β3. These were accompanied by the reduced expression of CTGF, fewer α-SMA
positive myofibroblasts, and less deposition of collagen in the healed neodermis,
consistent with less scar formation. In addition, by using a DEB mouse skin
transplantation model, we found that topically applied rC7 into transplanted DEB skin
not only restored C7 expression and AFs at BMZ but also corrected dermal-epidermal
separation of transplanted DEB skin. This study provides the first evidence to potential
use topically applied rC7 in improving chronic wound healing, relieving symptoms in
DEB, and providing better quality of life to patients who are suffering from chronic
cutaneous wounds.
37
3.2 Results
3.2.1 Topical rC7 incorporated into the regenerated BMZ
We used purified rC7 to evaluate the feasibility of topical application for healing
of skin wounds and RDEB treatments. Chen’s lab has the ability to purify milligram
quantities of rC7 from the condition medium of gene corrected RDEB fibroblasts [1, 2].
We topically applied 30μg rC7 on 1cm
2
full thickness skin excision on the center of
dorsal in athymic nude mice (n=20), rC7 was equally mixed with a 10%
carboxymethycellulose vehicle gel. Healed wound skin biopsies were obtained at various
time points after the topical application, subject the samples to immunofluorescence
staining with the antibody specific for human C7. In the Figure 3.1a, the wounds applied
with rC7, showed that rC7 has been incorporated into the BMZ of the healed skin wounds
at 14 days, and kept persistent for at least 6 more weeks with one time application.
Wounds treated with vehicle (n=20) lacked human C7 at the BMZ. However, we didn’t
detect NC1 staining on the wounds, which are treated with NC1 domain of C7 (n=10). It
suggests that full length C7 is required to incorporate into the BMZ. This set experiment
supported that topically applied rC7 on full thickness wound was able to incorporate into
BMZ and persist even after the wounds healed.
Double labeling was also utilized to confirm that the human rC7 located at the
BMZ of the healed mouse skin. With a polyclonal antibody that recognizes both human
and mouse C7 and a monoclonal antibody that only recognizes human C7, as shown in
Figure 3.1b, the merged images proved the co-localization, meanwhile, the vehicle alone
treated samples showing the presence of mouse C7 only.
After confirmed that the ability of the topical application of rC7 could incorporate
into the mouse BMZ, we would like to find the minimal dosage of rC7 is required for
BMZ deposition. As shown in Figure 3.1c, 8, 16, or 32μg of rC7 is applied to the
wounds topically, and we observed the dose dependent increase in the incorporation of
rC7 into the mouse’s BMZ.
We were puzzled about whether rC7 goes to experimental animals’ circulation
38
since it has been applied in full thickness wounds. To test it, we examined the sera
obtained from treated mice at four time points by immunoblot analysis. We did not
detect any rC7 in the sera at 6h, 24h, 48h and 1-week time points, with detection as low
as 10ng rC7 running as the control, see Figure 3.1d.
3.2.2 Topical rC7 promoted wound healing via re-epithelialization of the epidermis
After demonstrated topically applied C7 incorporated into the BMZ of healed
wounds, the next step is to determine whether topically applied rC7 could accelerate the
skin wound healing. The entire wound healing process was photographed from the
wounding day 0 until day 14, and the rC7 treated wounds exhibited remarkable
acceleration while comparing with vehicle treated wounds, as shown in Figure 3.2a and
3.2b.
Chen’s lab previously found that C7 promotes human keratinocytes migration in
vitro [3]. Re-epithelialization is a critical step for expediting wound healing in vivo,
which mainly depends on keratinocytes migration. To investigate whether the enhanced
wound healing in C7 treated wounds is due to faster keratinocytes migration in vivo, we
excised the full thickness wound with surrounding normal tissue at day 7 and subjected
the tissue to H&E staining. With the help of light microscopy, we measured the
epidermal gap length on the wound, and calculated the percentage of current un-re-
epithelized open wound length compared to original wound length. As shown in Figure
3.2c, the epidermal gap of rC7 treated wound is significant reduced compared to vehicle
treated wounds. The dotted yellow lines exhibited the rC7 treated wounds having more
re-epithelialization than vehicle treated wounds. Also, we enlarged the re-epithelializing
tongue (ReT) to declare the re-epithelialization edge.
We performed immunohistochemistry (IHC) staining to confirm the presence of
keratinocytes on re-epithelializing tongue and to determine the existence of blood vessels
in wounds. By using anti-pan keratin antibody, we observed the keratinocytes staining
on both ReT of rC7 and vehicle treated wounds; with anti-PECAM-1 antibody, we also
saw the endothelial cells in the wound bed, and there is no quantity differences between
rC7 and vehicle treated wounds.
39
Re-epithelialization is not only depending on the speed of keratinocytes migration,
but also about epithelial cells proliferating rate. With the question whether rC7 affects
the cell proliferating rate, we performed IHC staining on biopsy specimens with an
antibody specific to proliferate cell nuclear antigen (PCNA), which indicated the cell
proliferation speed. As shown in Figure 3.2d, we didn’t see meaningful differences
between these two treatments. Also, we compared the thickness of the epidermis during
wound healing process, healing wounds has been harvested at day 19 for both treatments.
There was no significant difference observed in the thickness of the epidermis, either rC7
or vehicle treated wounds at each time point. Based on these data, we believe that the
acceleration of wound healing by rC7 is more likely due to the boost of the re-
epithelialization, other than promoted wound angiogenesis or cell proliferation.
Although we stated that the non-collagenous NC1 domain of C7 is not capable to
enhance keratinocyte migration in vitro [3]. However, it is critical to check whether it’s
responsible for full-length rC7 promoting wound healing in vivo. We applied the same
molar concentration of NC1 domain as full-length rC7 in the topical wound healing
experiments. At days 9, 12 and 14, we observed the similar wound healing speed while
comparing the NC1 domain and vehicle treated mice, results shown in Figure 3.3a and
3.3b.
All data taken together, we believe that full-length rC7 can promote the wound
healing process is directly related to its ability to enhance keratinocytes migration.
3.2.3 Topical rC7 inhibited fibrosis of the wounded skin
During our wound healing experiments, we consistently observed that rC7 treated
mice presented less scarred wounds. Scarring is an outcome of wound healing, which
happens in the contraction phase. To demonstrate whether rC7 can reduce the
contraction, we conducted fibroblast-populated collagen I lattice contraction assay
(FPCL), a functional assay that measures collagen contraction in vitro, with and without
presence of full-length rC7 or its NC1 domain [4]. As we predicted, the presence of
serum growth factors resulted in the maximum contraction, while no contraction was
observed without the serum growth factors, shown in Figure 3.4a and 3.4b. Only full-
40
length rC7 showed the inhibition of contraction of the FPCL, but not the NC1 domain.
Similar as we showed before that the NC1 domain was not able to promote skin cells
migration in vitro or promote wound healing in vivo. Additional, rC7 inhibited of
collagen contraction assay in a dose dependent fashion with maximum level of inhibition
at 25 μg/ml. These data suggest that the domain of rC7 that is responding to accelerate
migration is also the domain that promotes wound healing in vitro.
During the wound healing process, a part of dermal fibroblasts will transform into
α-smooth muscle actin (α-SMA) positive myofibroblasts, which is responsible for
converting granulation tissue into permanent scars. There is a positive association
between the level of α-SMA and the fibrosis potential of healing wounds [5]. Therefore,
to determine whether rC7 could inhibits fibrosis in vivo, we tried to detect the level of α-
SMA with rC7 or vehicle treated skin wounds two weeks after topical application by
immunohistochemistry. As the Figure 3.4c shown, a dramatic decrease in α-SMA
positive myofibroblasts was observed of in rC7 treated wounds versus vehicle treated
wounds.
TGF-β is a secreted growth factor highly involved in all phases of wound healing.
The transformation of fibroblasts to myofibroblasts is induced by TGF-β, and the
connective tissue growth factor (CTGF) is one of the important mediate molecules [6].
As shown in Figure 3.4c, the rC7 treated wound also has a reduced expression of CTGF
in comparison with vehicle treated wounds after two weeks of treatment.
We also performed Picrosirius red and Masson’s trichrome stain to label the
collagen on the healed wounds. Compared to the vehicle treated wound, from Figure
3.4d, we observed that rC7 treated wounds not only had less dermal collagen deposition,
but also revealed more organized collagen fiber arranged in a parallel fashion.
With a significant reduction of α-SMA and CTGF expression, plus the less and
better collagen fiber rearrangement in rC7 treated full thickness skin wounds, these in
vivo data suggests that rC7 could inhibit scar formation.
In the inflammation phase of wound healing, TGF-β plays a vital role in
41
recruiting anti-inflammatory cytokines to clear the inflammatory necrotic tissue in the
newly wounded area [7]. However, excessive TGF-β can causes scar formation in the
remodeling phase. TGF-β has three isoforms, TGF-β1, TGF-β2, and TGF-β3. TGF-β1
and TGF-β2 isoforms display pro-scarring properties, while TGF-β3 isoform exhibits
anti-scarring property [8]. To determine whether topical applied rC7 effects the
expressions of TGF-β isoforms, we conducted immunostaining on healed skin wounds
with antibodies specific to each TGF-β isoform. As shown in Figure 3.4e, comparing
rC7 treated wound to vehicle treated wound, the expression of anti-fibrogenic TGF-β3
was significant increased, while the expression of pro-fibrogenic TGF-β2 was reduced.
We did not detect the expression differences of TGF-β1 between rC7 treated and vehicle
treated wounds (data not shown). These data suggest that one possible mechanism of rC7
is to reduce the scar formation by regulating the expression of TGF-β isoforms.
With the encouraging results from murine skin, we also tried to determine the
effect of rC7 on wound healing in human skin graft. This was done by grafting
1.5×1.5cm pieces of normal human skin onto the dorsal skin of athymic nude mice as
previously described [9]. We punch biopsied 8mm
wound from the normal human skin
grafts after they been grafting on mice for eight weeks, then topically applied either 30μg
of rC7 or equal volume of vehicle only. As shown in Figure 3.5, from photographs at
different time points, rC7 treated normal human skin wounds healed much faster than
vehicle treated wounds.
Collectively, the data above demonstrated that topically applied rC7 can enhance
the speed of skin wound healing in both murine and normal human skin graft models.
3.2.4 Topical rC7 restored C7 expression in engrafted RDEB mouse skin in vivo
RDEB patients are living with widespread and persistent wounds, where chronic
wound healing throughout the majority of their lives. To assess the effects of topical rC7
application on RDEB wounds, we transplanted the dorsal skin from C7-knockout mice
onto the dorsal side of athymic nude mice. There is no AF in the RDEB mouse since
they do not have C7 at the BMZ of skin [10]. The RDEB mouse model demonstrated the
most severe RDEB patients’ phenotype in clinical, genetic, and ultra-structural properties.
42
They developed blisters and erosions at or shortly after being born, and usually died
within a week. Due to the nature of the RDEB mouse model, which was not technically
feasible to perform wound-healing procedure on it. Alternatively, we scarified and
transplanted RDEB mice dorsal skin onto the backs of athymic nude mice. Subsequently,
we punch biopsied a 6mm wound over the grafted skin, and topically applied rC7. In
panel A-B of Figure 3.6a, DEB mouse skin exhibited the separation and no C7
expression at BMZ before transplantation, ensuring the characteristics of its origin.
However, after two weeks, the separations of epidermal and dermal junction were
diminished in the rC7 treated wounds, and had C7 restored at the BMZ.
DEB mouse skin wounds are usually superficial bullous type, which occurs at the
epidermal and dermal interfaces simultaneously. We debated whether topically applied
rC7 still could incorporate into the BMZ without wounding the engrafted skin. As shown
in panel D of Figure 3.6a, the separation of dermal epidermal junction still occurred in
the rC7 treated grafted skin, without detectable C7 by immunofluorescence staining.
These data indicates that wounding is a prerequisite for topically applied rC7 to
incorporate into the BMZ.
Immuno-EM results in Figure 3.6b disclosed that topically applied rC7 restored
the formation of AFs in the grafted DEB mouse skin, implying correction of the vital
ultra-structural disease abnormality.
Taken together, these data suggest that topical application of rC7 could restore the
C7 expression at BMZ and subsequently correcting the defects of RDEB skin by
enhancing the adherence and forming the AFs.
3.3 Discussion
In this chapter, we evaluated the feasibility of topical application with rC7 in
treating normal and RDEB wound healing, which were done by using an athymic nude
murine wound healing model, and normal human skin and DEB mouse skin transplant
models. Results of the study indicated that topically applied rC7 accelerated wound
healing by enhance re-epithelialization and incorporated into the BMZ of the healing
43
wounds. Moreover, we found that rC7 mediated wound healing also accompanied the
decreased expression of α-SMA and CTGF, which are indicating less scar formation.
Furthermore, our results indicated the correlation between the domain of C7 that
promotes keratinocyte migration in vitro and the domain of C7 that accelerates wound
closure in vivo. Lastly, we also demonstrated that topical applied rC7 incorporated into
the BMZ of DEB mouse skin in its transplantation model, formed AFs and corrected the
RDEB clinical phenotype.
It has been identified that plentiful growth factors and other agents could enhance
tissue regeneration; however, for various reasons their therapeutic application has been
limited in clinical medicine. The most common ones are lack of retention of the
therapeutic agents, poorly penetrating tissue, and instability of the agents in the protease
rich environment of a healing wound. With these reasons in mind, the first and only FDA
approved PDGF therapy, Regranex, only has a modest effect on wound healing and needs
to be administrated daily. The natural characteristics of C7 may provide advantages for
its use as a wound-healing agent. The molecular weight of C7 is 900 kDa, it is stable,
and relatively resistant to proteases. We tested the stability of purified rC7 by leaving it
in neutral buffer at room temperature for 3 months or at 4 °C for 6 months, and found no
degradation or loss of bioactivity [11].
We demonstrated that topically applied rC7 stably incorporated into the mouse
BMZ for up to 2 months as the treatment for RDEB. The actual turnover time of C7 in
human skin remains unknown. Our previous study showed that rC7 endures for at least 3
months in a human RDEB skin equivalent model [2]. In fact, only 35% of normal AF
complement is needed to maintain the integrity of skin in human. Regarding RDEB
patients, it’s possible that periodic topical applications of rC7 can improve their fragile
skin and prevent the formations of erosions and blisters [12]. The detailed administration
method is to be determined in future clinical trials.
Chen’s lab has developed three different administration methods to apply rC7 to
RDEB patients; they are intradermal injection, intravenous infusion, and topical
application. In the intradermal injection study, we showed that 20μg of rC7 was
44
sufficient to restore C7 expression at the BMZ, and reverse the RDEB phenotypes in a
RDEB skin equivalent model [13]. With the intravenous infusion approach, the
corrections of RDEB clinical characteristics were achieved with 60μg rC7 in a RDEB
skin transplantation model [14]. In this topical application study, 30μg rC7 was used to
alleviate the clinical symptoms of RDEB. It was observed that intradermal and topical
applications required less amount of rC7 to function compared to intravenous infusion.
Nevertheless, without a parallel systematic study to compare these three approaches, we
are unable to confirm which rC7 application will be the most efficient and efficacious in
clinical using.
In the study of intravenously infusion of C7, we exhibited that injected C7 over
expressed RDEB fibroblasts accelerated the wound healing speed compared with control
mice injected with uncorrected RDEB fibroblasts [15]. And in this study, we
demonstrated that topically applied rC7 could speed up the wound closure. The
mechanism(s) of how rC7 promotes wound healing remains unknown. The wound
healing process involves a series complicated biological phases; they are synchronized
happenings and overlap with each other, including inflammation, epithelialization,
granulation tissue formation, neovascularization, wound contraction and extracellular
matrix reorganization [7]. The epithelialization starts with keratinocytes migration.
Once the skin is wounded, the keratinocytes start moving and contact ECM protein and
glycoproteins, such as fibronectin while migrating across to close the wound bed [16, 17].
We found that C7 has the highest pro-motility potential to drive human keratinocytes
migration compared to other ECM proteins in previous studies. C7 was detected in the
late event of wound healing process, and did not appear at the BMZ until 7 days after
wounding [18]. Therefore, the advantage of exogenously delivering rC7 to wound will
be providing a matrix in the early time, which promotes the keratinocytes migration and
closes the wound faster. Our data were consistent with this assumption.
This study indicated that rC7 inhibits collagen lattice contraction in vitro. In
addition to this, topically applied rC7 also could reduce the quantity of α-SMA positive
myofibroblasts, the expression of CTGF, and the disorganized collagen deposition in the
newly healed wound. Since α-SMA and CTGF are the key indicators in the fibrosis,
45
these data raised the question whether exogenous applying rC7 to skin wounds could
reduce the scar formation. Following are findings to support this hypothesis. Firstly,
RDEB patients who lack of functional C7 always heal with tremendous scars. Secondly,
in previous studies, over-active fibrotic processes in a hypomorphic DEB mouse model
have been found, which only expresses 10% of C7 level with a better survival rate and
mimic the clinical features in RDEB. Its fibrosis is accompanied with upregulations in
the TGF-β pathway, which includes TGF-β and CTGF. Furthermore, increased α-SMA
positive myofibroblasts and tenascin C have been observed in healed wounds of
hypomorphic DEB mice. Lastly, the first trimester of fetus wound healing in utero is
well known as “scarless wound healing” [20]. During fetal life, the fetus is living in
amniotic fluid and enwrapped with an amniotic membrane, which is rich of C7 and
laminin 332, two large ECM molecules that improve wound healing [21]. Nowadays,
amniotic membranes also have been applied topically on burn wounds, ocular surface
reconstruction and skin wounds to promote the re-epithelialization and reduce the
excessive fibrosis [22].
There are numerous soluble factors involved in wound repair. TGF β is one
important factor that has been considered in scar formation. The TGF β family members
are similar in structure, but could be distinct from each other in function. TGF β1 and
TGF β2 possess pro-fibrogenic activities, whereas TGF β3 acts as an anti-fibrogenic
factor [8]. In this chapter, we found that topically applied rC7 caused upregulation of
TGF β3, and downregulation of TGF β2. In addition, we also showed increasing
organized collagen deposition and reductions of fibrosis markers, α-SMA and CTGF.
Combining these findings, it is strongly suggested that rC7 mediated less scarring wound
healing process due to the regulation of the expression of TGF β family members.
To prevent possible increasing immune responses, for example, production of
anti-C7 antibodies after human rC7 applied topically, or rejection of the transplanted
human or DEB mouse skin, we utilized immunodeficient athymic null mice to conduct
experiments in this chapter. To confirm the rC7 mediated accelerated wound healing and
reduced expression of α-SMA and CTGF, we also repeated the experiments with
immunocompetent mice and observed similar outcomes.
46
Several therapeutic strategies are under investigation in the RDEB field globally.
Among these gene, cell and protein therapies, a low risk, dependable and effective
method is not yet available [23]. A handful “proof of principle” clinical studies have
been initiated, including bone marrow/stem cell therapy and intradermal injection of
fibroblasts. Bone marrow/stem cells have been approached to a limited number of RDEB
patients with some success, but also companied with inevitable mortality rate. The
intradermal injection of allogeneic human dermal fibroblasts applied to RDEB patients
has increased their intrinsic mutated C7 at the BMZ and improved dermal epidermal
adherence, though the injected fibroblasts did not last longer than two weeks in the skin
of RDEB patients [1, 2, 13, 24, 25]. Using two preclinical animal models, we found that
intradermal injection of rC7 protein, fibroblasts expressing C7, and lentivectors
expression C7 into DEB mice all restored C7 and AFs at the BMZ and significantly
prolonged the survival of DEB mice [14].
Compared to other delivery methods, administrate rC7 topically would have
various advantages. First, no concerns of exposing patients to exogenous DNA, RNA,
viral vector, and live cell are present. Additionally, local application is safer and more
durable than any intravenous administrations. Topical application of rC7 would be a
simple and painless administration method, which helps RDEB patients by improving
current wound healing and preventing the potential blistering. Nevertheless, by
thoroughly considering the condition of RDEB patients, we also realized that this method
is not capable to reach and treat esophageal and alimentary tract lesions, which are
commonly in the most severe RDEB patients. Also, topically applied rC7 can only be
applied to open wounds, so it could not penetrate the integrate skin or prevent the onset
of coming skin blisters. Lastly, similarly to other protein replacement delivery methods,
topical application of rC7 requires lifetime continual treatments.
In conclusion, our studies demonstrate that topically applied rC7 could accelerate
wound closure, stably incorporate into the newly regenerated BMZ of skin wounds,
improve the quality of healed wound by reducing excessive scars. Furthermore,
considered as a treatment for general chronic skin wounds, this method could largely
benefit the RDEB patients who have consistent opening wounds and often develop
47
metastatic squamous cell carcinoma leads by perennial chronic skin wounds. This study
does not only provides a promising treatment option for general skin wounds, but also
improves life quality and prolong the life span for RDEB patients as well. With all the
encouraging data above, disclosing the mechanism of how rC7 functions in wound
healing and conducting efficacy and safety studies will be the next steps of our journey.
3.4 Material and Methods
Purification of rC7
Human rC7 was purified from serum-free media from RDEB dermal fibroblasts
stably transduced with a lentiviral vector coding for full-length C7 as described [2].
Briefly, serum-free media were equilibrated to 5 mmol/l EDTA, 50 μmol/l PMSF, and 50
μmol/l NEM and precipitated with 300 mg/ml ammonium sulfate at 4 °C overnight with
constant stirring. Precipitated proteins were collected by centrifuging at 1.2 × 10
6
g/minute for 1 hour, resuspended, and dialyzed in Buffer A (65 mmol/l NaCl, 25 mmol/l
Tris–HCl, pH 7.8). Following dialysis, insoluble material was collected by centrifugation
at 8,600g for 20 minute, and the pellet redissolved in Buffer B (50 mmol/l Tris–HCl pH
7.5, 150 mmol/l NaCl, 5 mmol/l EDTA, 2 mmol/l NEM, 2 mmol/l PMSF). The solution
was clarified as above, and the supernatant, S1’, was passed over a Q-sepharose column
(Pharmacia, Piscataway, NJ) equilibrated in the same buffer. Elution was then carried
out with a linear gradient from 0.2 to 1.0 mol/l NaCl of appropriate volume size. The rC7
eluted at 0.7–1 mol/l NaCl. The NC1 domain of C7 was purified from 293 cells stably
transfected with cDNA encoding for NC1 as described [26].
Topical application of rC7 onto mice
We first made a 1.0×1.0cm full thickness excisional wound by lifting the skin
with forceps and removing full thickness skin with a scissors on the mid-back of 8- to 10-
week-old athymic nude mice (Simonsen Laboratory, Gilroy, CA). Immediately after
wounding, 8–32 μg of rC7 or 30 μg NC1 in a 10% carboxymethylcellu- lose gel were
applied to the wound surface. We then covered the wound with a band-aid and a Coban
self-adherent wrap, to prevent desiccation. We treated 30 mice with topical rC7, 10 mice
48
with NC1, and 20 mice with the vehicle as a negative control. 2 to 8 weeks after topical
application, biopsies from the wounds were obtained and subjected to immunostaining
using an antibody specific for human C7 (clone LH 7.2; Sigma, St Louis, MO) or a rabbit
polyclonal antibody that recognizes both human and mouse C7 [27]. To measure the
wound size, standardized digital photographs were taken of the wounds at various days
after wounding and open wound areas determined with an image analyzer (AlphaEase FC
version 4.1.0; Alpha Innotech, Johannesburg, South Africa) on a personal Macintosh
computer. The total pixels that covered the unhealed areas were drawn onto the digital
photographs using a pattern overlay in ImageJ (http://rsbweb. nih.gov/ij/). The number
of pixels covering an open wound area on a given day was divided by the number of
pixels spreading over the initial wound on day 0 to obtain the percentage of closure.
For RDEB mouse skin transplantation studies, 1.2 by 1.2cm of skin from 2 to 5
days old, newborn RDEB mice was transplanted onto the back of athymic nude mice. At
12–14 days after skin transplantation and engraftment, the RDEB skin grafts were either
unwounded or wounded with a 6 mm punch biopsy instrument. We then topically
applied 30 μg of rC7 (n = 15 mice) or vehicle (n = 4) to the wounded RDEB skin or
unwounded RDEB skin (n = 4) as described above.
For evaluating wound healing of human skin, a 1.5 × 1.5 cm square of full-
thickness human skin was grafted onto athymic nude mice as previously described [9].
Eight weeks after grafting the engrafted human skin was wounded using a 8mm punch
biopsy tool. We then topically applied rC7 (30 μg) or vehicle to the wounds and
bandaged as described above. The assessment of wound healing was then performed
using area planimetry, as described above for the murine wounds. All animal studies
were conducted using protocols approved by the University of Southern California
Institutional Animal Use Committee.
Immunofluorescence staining and ultrastructural analysis of tissue
Five-micrometer thick sections of OCT-embedded frozen tissues were cut on a
cryostat, fixed for 5 minutes in cold acetone, and air-dried. Immunolabeling of the tissue
was performed using standard immunofluorescence methods as described previously [1,
49
2, 13, 24]. Briefly, for single- and double-immunofluorescence staining, sections were
blocked with M.O.M. Mouse IgG Blocking Reagent (Vector Laboratories, Burlingame,
CA) for 1 hour at room temperature. Primary antibodies were diluted in phosphate
buffered saline with 1% bovine serum albumin. For C7 staining, we used monoclonal
antibodies against human C7, clone LH 7.2 (Sigma), or a rabbit poly- clonal antibody that
recognizes both mouse and human C7 [27]. For double- immunofluorescence staining,
we incubated the mouse monoclonal anti-human C7 antibody together with a rabbit
polyclonal antibody to both human and mouse C7. For TGF-staining, we used
polyclonal antibodies against TGF- 1 (sc-146, Santa Cruz Biotechnology, Santa Cruz,
CA), TGF- 2 (sc-90, Santa Cruz Biotechnology), or TGF- 3 (sc-82, Santa Cruz
Biotechnology). All primary antibody dilutions were 1:200. After incubation for 1 hour
at room temperature, sections were washed in phosphate buffered saline three times and
stained for 1 hour with FITC-conjugated goat anti-mouse IgG1 with or without Cy3-
conjugated goat anti-rabbit IgG (Sigma) diluted 1:300 in phosphate buffered saline with
1% bovine serum albumin. Slides were mounted with 40% glycerol. Photographs of
stained sections were taken using a Zeiss Axioplan fluorescence microscope equipped
with a Zeiss Axiocam MRM digital camera system (Carl Zeiss International, Göttingen,
Germany).
Immunogold electron microscopy was performed on the engrafted RDEB mouse
skin using a standardized method as described previously. To assess human AF
formation and ultrastructure, 40 micron sections were fixed in 0.1% glutaraldehyde,
rinsed in 0.15 mol/l Tris pH 7.5, then incubated in our polyclonal anti-NC1 antibody
followed by 5nm gold secondary antibody and enhancement as described [28, 29].
Histological analysis of tissues
The mice whose wounds were treated with topical rC7 or vehicle were euthanized
at 7 or 14 days after treatment. The wounds, together with unwounded skin margins,
were excised and put into 10% formaldehyde. H&E staining was carried out as
previously described [30]. To show the entire wound, multiple overlapping photographs
were taken under a microscope (Nikon, Eclipse TE2000-U, ×4; Nikon, Tokyo, Japan) and
50
used to reconstitute the entire wound. A standard immunohistochemistry staining
procedure was carried out as described [31]. We used a mouse monoclonal antibody to
pan keratin (Clone 80; Abcam, Cambridge, MA), a rabbit polyclonal antibody to
PECAM-1 (Clone M-20; Santa Cruz Biotechnology), a mouse monoclonal antibody to α-
SMA (Clone 1A4; Dako Denmark A/S, Glostrup, Denmark), a mouse monoclonal
antibody to PCNA (Clone PC10; EMD Millipore, Billerica, MA), and a rabbit polyclonal
antibody to CTGF (Abcam). All antibodies were used in 1:100 dilutions.
Fibroblast-populated collagen I lattice contraction assay (FPCL)
Type I collagen (Sigma) lattices were prepared as previously described[4].
Human dermal fibroblasts were cultured in Dulbecco’s modified Eagle’s medium
(Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum with antibiotics. The
lattices were prepared with a final fibroblast density of 100,000 cells/ml in a 0.6 ml
volume per well in 24-well, non-tissue culture plates (Becton Dickinson, Le Pont De
Claix, France). After polymerization of the collagen (VitrogenTM, Cohesion
Technologies, Palo Alto, CA), the lattices were incubated in 1.0 ml Dulbecco’s modified
Eagle’s medium with or without fetal bovine serum. Either NC1 or rC7 was added to the
individual dishes at concentrations ranging from 6.25–30 μg. The collagen lattices were
incubated at 37 °C with 5% CO
2
and the contraction of the lattices was measured by
weight.
24
All experiments were carried out in triplicate and repeated three times, and data
points and error bars in the figures represent averages and standard deviation.
51
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15. Woodley, D.T., et al., Intravenously injected human fibroblasts home to skin
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53
Figures:
Figure 3.1 Topically applied rC7 stably
incorporated in the regenerated BMZ in the
mouse skin.
(a) Immunofluorescence staining of the mouse
skin was performed with an antibody specific
for human C7 at 2 weeks after the topical
application of 30 μg of rC7, NC1, or vehicle
alone. Note that the healed wounds treated
with rC7 (n = 30 mice) demonstrated a linear
pattern of C7 deposition at the BMZ (panel A).
In contrast, no human C7 was detected in mice treated with vehicle alone (n = 20 mice)
or NC1 (n = 10 mice) (panels B and C). Panel D shows the stable incorporation of
human rC7 at the mouse BMZ
at 8 weeks after the initial
topical application of rC7.
(b) Immunofluorescence
staining of mouse skin was
performed 2 weeks after the
topical application of rC7. The
skin sections were labeled with
either a monoclonal antibody
specific for human C7 (green,
panel -H) or a rabbit
polyclonal antibody that
recognizes both mouse and
human C7 (red, panel -M+H).
Merged images demonstrate
colocalization of topically
applied human rC7 with endogenous mouse C7 at the mouse BMZ. The lower panel
depicts staining of wounds treated with the vehicle (VE) alone. (c) Dose-dependent
deposition of human rC7 at the mouse BMZ after topical rC7 application.
Immunofluorescence staining of biopsy specimens were performed with an antibody
specific for human C7 after the animal’s wounds were treated with 8 μg (A), 16 μg (B),
or 32 μg (C) of topical rC7, respectively. Scale bar: 200 μm.
54
Figure 3.1 Topically applied rC7 stably
incorporated in the regenerated BMZ in the
mouse skin. (d) Sera were taken from mice at the
time indicated after topically applied with 30 μg
rC7 and subjected to 4–15% SDS-PAGE followed
by immunoblot analysis using an anti-NC1
antibody. Purified rC7 of 10 ng was run as a
control (Con). The positions of full-length 290
kDa C7, 50 kDa mouse IgG heavy chain (mIgGH),
and molecular weight markers are indicated. d,
dermis; e, epidermis.
Figure 3.2 Topical application of rC7 promoted wound healing. 1.0 cm
2
(1×1cm)
square full-thickness excision wound was made on the central dorsal of 8 to 10 week-old
athymic nude mice, and rC7 (30 g) was applied topically once on day 0 (n = 20 mice per
group). (a) Representative days 0, 7, 11, and 14 wounds are shown. (b) Mean ± SD
open wound area measurements at days 0, 7, 9, 11, and 14 after wounding (n = 20 mice
for each group).
55
Figure 3.2 Topical application of rC7 promoted wound healing. (c) On day 7,
excisional biopsies of full-thickness wounded skin with a portion of unwounded skin
were obtained from wounds treated with topical vehicle (VE) or rC7 (C7). The biopsy
specimens were stained by H&E and photographed with a light microscope.
Independently, photo- graphed images with identical magnifications were reconstituted to
show the unhealed areas of the wounds. Red dotted lines indicate the unhealed wound
area. Yellow dotted lines mark the newly re-epithelialized epidermis. The fronts of
newly re-epithelialized epidermis were enlarged, as shown in higher magnified images.
Scale bars: 0.33mm.
56
Figure 3.2 Topical application of rC7 promoted wound healing. (d)
Immunohistochemistry analysis of biopsy specimens of day 7 full-thickness wounds
treated with either vehicle (VE) or rC7 (C7) with anti-pan keratin (keratinocytes), anti-
PECAM-1 (endothelial cells), and anti-PCNA antibodies. Ten randomly selected images
per each condition from three independent experiments were analyzed for consensus.
Representative images are shown. Scale bars: 0.3 mm (left column); 0.2 mm (middle and
right columns). In the left column, arrows point to the keratin-labeled, re-epithelializing
tongue (ReT); in the middle column, the arrows point to blood vessels; in the right
column, the circles point to proliferating keratinocytes.
57
Figure 3.3 Topical application of NC1 did not promote wound healing. A 1.0 cm
2
(1
× 1 cm) square full-thickness excision wounds were made on the mid-back of 8 to 10
weeks old athymic nude mice and purified NC1 domain (30g) (n = 10 mice) or vehicle
(VE) (n = 10 mice) were applied topically once on day 0. (a) Representative days 0, 7,
12, and 14 wounds are shown for wounds treated with NC1 or vehicle alone (VE). (b)
Mean ± SD wound size measurements at days 0, 7, 9, 12, and 14 after wounding treated
with NC1 (n = 10 mice for each group).
Figure 3.4 C7 inhibited the contraction of collagen lattices in vitro and reduces the
presence of myofibroblasts, CTGF expression, and collagen deposition in vivo. (a)
Early passage, human dermal fibroblasts were isolated and mixed with serum and type I
collagen. Collagen lattices, with size of 0.6cm
3
and cell density at 100,000 cells/cm
3
,
were released and floated in 1ml DMEM. rC7 or NC1 of 30 μg were added to the serum
containing medium and incubated at 37 °C for 24 hours. Contraction assays were also
carried out without serum added (GF−) or with serum added (GF+). Contraction of the
58
lattices was determined by measuring the gel weight. These data represent the mean ±
SD of triplicate determinations in one representative experiment. Similar results were
obtained in two other independent experiments. (b) Dose-dependent inhibition of
contraction of collagen lattices by rC7. rC7 at the indicated concentrations were added to
the contraction assays. These data represent the mean ± SD of triplicate determinations
in one representative experiment. Similar results were obtained in two other independent
experiments.
(c) Immunohistochemistry staining of healed mouse skin was performed with the
antibodies specific for α-SMA and CTGF at 2 weeks after topical application of vehicle
(VE) and rC7. Ten randomly selected images per condition from three independent
experiments were analyzed. The representative images are shown. Scale bars: 0.2mm.
Note that the wounds treated with topical rC7 showed significantly diminished α-SMA–
positive fibroblasts (myofibroblasts) as well as decreased CTGF expression. (d)
Collagen deposition was assessed by Masson’s trichrome staining and Picrosirius red
staining of healed wounds at 14 days after topical application of rC7 or vehicle (VE).
Ten randomly selected images per condition from three independent experiments were
analyzed. The representative images are shown. Scale bars: 0.2 mm. Note that rC7
treated wounds have less collagen deposition in the dermis by both staining methods
compared with vehicle-treated wounds, which show perturbed fiber architecture. (e)
Immunofluorescence staining of healed mouse skin was performed with the polyclonal
antibodies specific for TGF-β2 and TGF-β3 at 2 weeks after topical application of VE
and rC7. Ten totally randomly selected images per condition from three independent
experiments were analyzed. The representative images are shown. Scale bars: 0.2mm.
Note that the wounds treated with topical rC7 display reduced expression of TGF-β2 and
increased expression of TGF-β3.
59
Figure 3.5 Topically applied rC7
promoted wound closure of human
skin. A 0.5 cm
2
(8 mm diameter
punch biopsy) full-thickness excision
wound was made in human skin
grafted onto the mid-back of 8 to 10
weeks old athymic nude mice. The
wounds were then treated with
topically applied 30μg rC7 (C7) or
vehicle (VE) (n = 5 mice per group).
(a) Representative days 0, 5, 7, and 10
wounds are shown. Wound sizes were
reduced in wounds treated with rC7
compared with vehicle control at 5, 7,
and 10 days after wounding. (b) Mean
+ SD wound size measurements at
days 0, 5, 7, 10, 12, and 14 after
wounding (n = 5 mice for each group).
Figure 3.6 Topical application of rC7 incorporated into the BMZ of RDEB mouse
skin grafts and forms AFs in vivo. (a) Histological appearance (A–D) and
immunofluorescence staining (E–H) of engrafted RDEB mouse skin using a polyclonal
antibody to the NC1 domain of C7. Panels A and E, 2 weeks after grafting of RDEB
mouse skin and before treatment (n = 30 mice); panels B and F, wounded and engrafted
RDEB skin 2 weeks after topi- cally applied vehicle (n = 4 mice); panels C and G,
wounded and engrafted RDEB skin 2 weeks after 30 μg of rC7 was applied topically (n =
15 mice); panels D and H, unwounded engrafted RDEB skin 2 weeks after 30 μg of rC7
was applied topically (n = 4 mice). (b) Immunogold labeling of engrafted murine RDEB
skin topically applied with 30 μg rC7 (C7) or vehicle (VE) was performed using an anti-
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NC1 polyclonal antibody and revealed that the topical rC7 incorporated into the RDEB
skin grafts and formed AFs. Note restoration of numerous arching AFs depicted with
arrows and labeled with gold particles decorating the BMZ in rC7-treated RDEB skin
grafts.
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Chapter IV
The Absence of Functional C7 in RDEB Causes Faulty Regulation of TGF β and
Profound Skin Scarring
4.1 Summary
In the last chapter of my thesis, I was aiming to reveal the mechanisms of how C7
functions in reducing the scar formation in skin wound healing by utilizing the human
samples from RDEB patients.
RDEB patients are having open wounds, bullous wounds and erosions through
their lives that caused by inherited skin fragility. Moreover, excessive scarring caused
fibrotic fusion of digits, joints contractures of limbs, the conglutination of lips, fixed
tongue and more other problems are also accompanied with RDEB patients. We already
observed topical applied C7 could accelerate wound closure and reduce excessive scars
by improving the quality of healed wounds in normal mouse skin model.
Dr. Woodley and Dr. Chen are conducting intradermal injection of human rC7
into RDEB patients clinical trial project since 2011. During the screening the potential
RDEB patients for our trial, we obtained skin and serum samples and initiated cell
cultures from twenty-two diverse conditioned RDEB patients. By characterizing skin
tissue, major skin cells including keratinocytes and fibroblasts and serum samples from
those patients, we discovered that RDEB patients display increased expression of pro-
fibrotic TGF β isoforms TGF-β1 and 2 as well as markers associated fibrosis. With
siRNA knockdown C7 in NFB and supplementary of rC7 to RDEB patients’ cells, we
confirmed that C7 loss is directly related to up-regulated TGF β signaling associated with
fibrosis. In addition, by suppressing TGF β signaling in RDEB patients’ primary
fibroblasts, we also retrieved fibrotic markers to normal expression level. The results
from this chapter could be the first evidence that discloses the mechanism of how
excessive scarring commonly existing in RDEB patients, and may provide a therapeutical
method to reduce the scarring that often leads to development of aggressive skin
squamous cell carcinoma and improve the life quality of RDEB patients.
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4.2 Results
4.2.1 Pre-clinical patient selection and characterization for phase I/II trial
In a previous study of intradermal injection of human rC7 and other required
studies by FDA, in order to find a potential treatment, we started to screen potential
candidates for a phase I/II trial to test intradermal injection of rC7 into RDEB patients [1].
The study was approved by the University of Southern California Institutional Review
Boards and was conducted according to the Declaration of Helsinki Principles.
Twenty-two voluntary RDEB patients were recruited from the United States, and
7 of them were under 18 years old. Based on the designs of the trial, we obtained skin
and serum samples from each patient to determine their disease clinically, pathologically,
ultra-structurally, and immunologically. An approximately 5mm x 8mm shave skin
biopsy was harvest from “normal looking” skin close to the wounds to determine the
expression level of endogenous C7 by an anti-NC1 antibody, the status of AFs by
transmission EM, the presence of anti-C7 autoantibodies at the BMZ by direct
immunofluorescence staining. We also isolated keratinocytes and fibroblasts from skin
biopsies and used these cells to study the molecular mechanism on why RDEB wounds
heal with excessive scars [2, 3]. We used patients’ sera to evaluate the presence of
autoantibodies to C7 by ELISA and identify the C7 gene mutation.
As seen in Table 4.1, we addressed the level of C7 expression at the DEJ of their
skin by immunofluorescence staining with an anti-NC1 polyclonal antibody. Nine RDEB
patients expressed reduced C7 compared to normal human skin, four of them showed the
absence of C7 in skin biopsies, and the remaining nine patients exhibited the same
expression level of C7 as normal human skin. The conditions of AFs were evaluated by
transmission EM for density and morphology. The results are highly correlated between
immunofluorescence and transmission EM. The patients who lack AF structural also
show a lack of C7 expression; among the remaining patients, the observed AFs appeared
either attenuated in quantity or abnormal morphology, and sometimes both. We also
tried to detect whether the patients have anti-C7 autoantibodies deposited onto BMZ. As
seen in Table 1, direct immunofluorescence staining on patients’ skin indicated that none
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of the RDMB patients appeared to have autoantibody deposited on BMZ.
To determine whether RDEB patients have anti-C7 antibodies, we subjected sera
to a commercially available ELISA utilizing NC1 and NC2 domains as the target
substrates. We used 13 EBA patients’ sera as positive controls and 17 normal subjects’
sera as negative controls to establish the threshold for the assay [4]. With the commercial
ELISA, 7 of 22 RDEB patient sera exhibited reactivity with values above the threshold.
To determine whether RDEB sera recognize C7 in the BMZ of skin, we performed
indirect immunofluorescence staining using salt-split human skin as substrate. None of
the sera of 22 patients bound to C7 on the dermal side of salt-split skin including those
seven RDEB patients who had autoantibody specific to C7 as evaluated by ELISA.
Taken the results of three assays into account, our data suggest that the anti-C7 antibodies
in their sera are unlikely pathogenic.
4.2.2 RDEB Patients display excessive scarring by up-regulated TGF β pathway
As discussed in the previous chapter, topical rC7 inhibited fibrosis of the full
thickness wounded mouse skin. Compared to vehicle treated healed wounds, human rC7
treated wounds exhibited reduced pro-fibrotic TGF-β2, increased anti-fibrotic TGF-β3,
and less α-SMA positive myofibroblasts in the dermis. Moreover, down-regulated TGF-
β2 also affected its downstream CTGF, a marker that indicated fibrosis, also exhibited a
decreasing trend. These were the pieces of evidences suggest that C7 may inhibit fibrosis
and excess skin scarring via TGF β signaling pathways [5]. TGF β signaling pathways
have been known that highly involved in the whole process of skin wound healing in
various aspects, and act in different roles. Such as during the phase of inflammations,
released TGF β isoforms recruit more growth factors to accelerate the speed of re-
epithelialization. In the phase of wound contraction and extracellular matrix
reorganization, excessive profibrogenic TGF β isoforms can lead the healing wound to
form a pathological scar [6].
We chose 4 of 22 patients based on disease severity distribution and the sample
availability; the four patients covered all three RDEB subtypes that presented a series of
different severity of the disease. Their C7 expressions are various and related to the
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severity of RDEB patients. From RDEB generalized, severe; RDEB generalized other;
and RDEB inversa, C7 expression is increasing as the pattern, and the severity is
reducing as expected [7].
As shown in Figure 4.1a, we performed immunofluorescence staining on patients
skin to determine the expression of TGF β isoforms and markers associated with fibrosis.
The results revealed significant increased expression of pro- fibrogenic TGF β isoforms,
TGF-β1 and TGF-β2. Consequently, the up-regulation of downstream canonical TGF β
pathway signaling, phospho-Smad2/3 (p-Smad 2/3), and extracellular matrices, C1, and
fibronectin. Although TGF-β1 and β2 both are pro-fiborgenic isoforms, the expression
pattern are unlike each other, TGF-β1 were up-regulated in either epidermis and/or
dermis, while TGF-β2 was dramatically up-regulated in the epidermis when compared to
normal human skin. The expression patterns of other downstream markers, p-Smad 2/3,
C1 and Fin were up-regulated in the dermis side. The bar graph in Figure 1b showed the
differences in ratio between patients and normal human subject in each molecule we tried
to detect.
In the Figure 4.1c-f, we also conducted immunoblotting analysis of primary skin
keratinocyte and fibroblast lysates from the four patients we selected, and compared the
levels of the expression of various markers with normal human cells. The results showed
up-regulation of TGF-β1, TGF-β2, p-Smad2/3, TGF- β RI in RDEB keratinocytes, and p-
Samd2/3, TGF-β RI, TGF-β RII, α-SMA, Fibronectin, and C1 in RDEB fibroblasts. The
results are consistent with results seen immunofluorescence staining performed on
patients’ skin. Using a commercial available TGF-β1 specific ELISA kit, we also found
the increased levels of TGF-β1 in the media of RDEB fibroblasts when compared with
normal human fibroblasts as shown in Figure 4.1g [8]. However, we did not detect the
differences in the levels of TGF-β2 in the media of fibroblasts between normal and
RDEB patients as assayed by a commercial available TGF-β2 specific ELISA kit (data
not shown). We also measured the level of TGF-β1 in the sera of RDEB patients and
normal control subjects using ELISA. As shown in Figure 4.1h, there are elevated levels
of TGF-β1 in the sera from all four RDEB patients compared with normal human sera.
65
Combining these exciting in vitro and in vivo results, which strongly indicated
that the loss of C7 in RDEB patients’ spurred pro-fibrotic TGF-β signaling expressions
and induced a distinct pro-fibrotic gene expression program. They may provide new
perspectives about the molecular mechanisms that underpin the development of fibrosis
and scarring in RDEB patients.
4.2.3 Loss of C7 direct related to up-regulated TGF β signaling
To evaluate if the RDEB patients’ excessive scars are caused by C7 loss that leads
to activation of TGF β fibrosis pathway, we conducted a series of experiments to test this
hypothesis, including (i) introducing C7 back to RDEB patients cells via a lentiviral
vector expressing COL7A1 [9]; (ii) direct adding rC7 back to cell culture medium; (iii)
silent the C7 expression on normal human fibroblasts by a pool of siRNAs specific to
COL7A1 [10].
We chose two RDEB patient’s fibroblasts cells that did not express any C7 and
exhibited significant up-regulations of TGF β fibrosis signaling pathway. We infected
these RDEB cells with our high titers of lentiviral vectors expressing C7. As shown in
Figure 4.2a, the C7 expression detected by immunoblotting 72 hours after transfection,
and the position of the band corresponding to full-length 290-kD C7 are indicated. For
the downstream signaling, we observed the down-regulation of p-Smad 2/3, α-SMA, C1,
and Fibronectin compared to the un-transduced cells. The ratio differences between
transduced and un-transduced shown in Figure 4.2b. The immunofluorescence staining
result from one of the patients’ fibroblast one week after transduction is shown in Figure
4.2c. The transduction efficacy is 50.8% compared with the un-transduced fibroblasts.
We also obtained the 48h incubated conditioned medium from transduced and un-
transduced RDEB patients’ fibroblasts and subjected them to TGF-β1 ELISA kit. The
bar graph in Figure 4.2d showed the reduced levels of the TGF-β1 in lentiviral
transduced RDEB fibroblasts when compared to un-transduced cells. Moreover, the
concentration of TGF-β1 in transduced RDEB cells was close to the conditioned medium
from normal human fibroblasts. The C7 lentiviral vector has also been used to transduce
RDEB patients’ keratinocytes. As shown in Figure 4.3a, The C7 expression is
66
detectable after 72h transduction by immunoblotting with an anti-NC1 antibody. As a
consequence of restored C7 expression in these cells, transduced cells exhibited
decreased expression of TGF-β1, TGF-β2, and p-Smad2/3 when compared to un-
transduced patients’ keratinocytes. β-actin was used as internal control to ensure the
equal loading. The bar graph in Figure 4.3b is shown the quantified ratio differences
prior and after the transduction. The immunofluorescence staining of one week
transduced cells showed that the transduction efficacy was 43.8% compared to the
normal human keratinocytes, as seen in Figure 4.3c.
Since C7 showed the high affinity to TGF-β1 in in vitro binding assay, to evaluate
if the binding of C7 with TGF β would prevent the TGF-β1 induced fibrosis signaling
pathway, we serum starved the cells and then stimulated the cells with TGF-β1 in the
presence or absence of NC1 or full-length rC7. As shown in Figure 4a, the up-regulated
expression exhibited in the TGF-β1 stimulated fibroblasts lysates in comparison to un-
stimulated cells. 40 μg/ml C7 or 20 μg/ml NC1 with 5ng/ml TGF-β1 were pre-incubated
on ice, followed by stimulating the serum starved human fibroblasts cultures for 15min at
37°C, and subjected harvested cell lysates to immunoblotting that showed down-
regulations of p-Smad 2/3 in the presence of C7 or NC1. And the changes in ratio are
shown in Figure 4.4b. For downstream fibrosis markers, we performed a similar
experiment; stimulated the serum starved human fibroblasts with 30 μg/ml C7 with
5ng/ml TGF-β1 for 48h, then harvested the cell lysates and performed immunoblotting
with antibody specific to α-SMA. As seen in Figure 4.4c, α-SMA expression decreased
20% in the rC7-contained samples when compared to the PBS treated negative control.
The 48h incubated medium also obtained and subjected to TGF-β1 ELISA kit, the result
in Figure 4.4d showed that TGF-β1 level decreased 30% in C7 contained sample when
compared to negative control.
We also tried to knock down C7 expression in normal human fibroblasts with a
pool of published (siRNA) [10]. As shown in Figure 4.5a, the C7 expression in normal
human fibroblasts has been knocked down completely 60h after transfection. The
expression level of TGF-β1 in 48h incubated condition medium exhibited up-regulation,
and the fibrogenic markers, C1 and α-SMA showed up-regulated expression in C7
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knocked down fibroblasts when compared to equal concentration scrabbled negative
siRNA transfected normal fibroblasts [10]. The bar graph in Figure 4.5b indicates the
expression changes of C7, C1 and α-SMA in ratio after considering the internal control
and corrected by Image J software [5]. Considering the possible variation in normal
human fibroblasts cell lines, we used two different batches of normal human fibroblasts
to perform the siRNA C7 knockdown experiment. TGF-β1 ELISA results of siRNA C7
knockdown in normal human fibroblasts shown in Figure 4.5c, secreted TGF-β1 levels
in both siRNA C7 knockdown normal human fibroblasts have increased 55%-70% when
compared to the same cell lines transfected by equal concentration of scrabbled negative
siRNA.
Based on the two approaches to re-introduce C7 to RDEB patients’ cells in vitro
and silent the C7 expression from normal human fibroblasts by siRNA, our data
demonstrated that the altered TGF β pro-fibrotic signaling in RDEB patients is caused by
the C7 loss. It furthermore indicates that the presence of C7 could inhibit excessive
scarring in RDEB patients.
4.2.4 Suppress TGF β signaling pathway in RDEB cells to retrieve fibrotic markers to
normal expression level
To validate whether the over-activated TGF β pathway is involved in the
mechanism of forming excessive scars in RDEB patients, we tried to: abolish the activity
of TGF-β1 with TGF β pan specific antibody, inhibit the signal transduction pathway
with chemical inhibitors, SB505124 to TGF-β R-I, and silent the key players of
downstream molecules by siRNA knockdown in canonical TGF β pathway.
As shown in Figure 4.6a, we performed immunoblotting on cell lysate prepared
from RDEB cells treated with 40 μg/ml TGF β pan specific antibody for 48h, and the
expression of α-SMA was dramatically decreased, as shown in Figure 4.6b. We also
obtained the medium to run TGF- β1 ELISA, as the bar graph shown in Figure 4.6c, the
TGF β1 signaling also reduced.
TGF-β R-I is one of the most important receptors to continue transducting the
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TGF β signaling to downstream, also named as ALK5. TGF-β R-I exhibited up-
regulation in RDEB patients skin cells by immunoblotting analysis. We utilized
chemical inhibitor SB505124, a reversible ATP competitive and selective ALK inhibitor
of ALK4 and ALK5, to block the transduction of TGF β signaling. As shown in the
immunoblotting results in Figure 4.7, the expression of α-SMA is decreased in the
RDEB fibroblasts incubated with 5 μM SB505124 for 48h. Since the inhibition of TGF β
R-I blocked the TGF β mediated downstream signaling, so the fibrosis marker, α-SMA
functions as a readout indicator also showed decreased expression. No cytotoxicity has
been observed at the experimental period and dosage.
Activated TGF-β signaling transduction either through canonical Smad dependent
pathway or non-canonical Smad independent pathway [11]. Several in vivo studies
suggested the significance of Smad in animal organs fibrogenesis. For example, it has
been shown that suppressing increased Smad 3 signaling could inhibit scar formation [12].
So we aimed to silent the receptor regulated Smads, Smad 2 and Smad 3 by siRNA
knockdown. As shown in the Figure 4.8a, we incubated RDEB patients’ fibroblasts with
a mixture of Smad 2 siRNAs or Smad 3 siRNAs for 60h, prepared cell lysates for
immunoblotting with antibodies specific to Smad2 or Smad3, and found that majority of
Smad 2/3 has been knocked down in the fibroblasts. As expected, fibrosis markers α-
SMA, C1 and fibronectin are up-regulated in the Smad2/3 knockdown RDEB patients’
fibroblasts when compared to the scrambled random siRNA transfected RDEB
fibroblasts [13]. The ratio differences are shown in Figure 4.8b bar graph.
We aimed at the molecules of TGF β pathway to minimize their activities by
neutralizing antibodies, inhibitor and siRNAs. The results of all approaches strongly
indicated that the up-regulated fibrotic markers could be attenuated once we deactivate or
suppress the TGF β signaling pathway.
4.3 Discussion
Over 700 gene mutations in RDEB patients have been published, however during
the screening RDEB patients for our ongoing clinical trial, we identified ten more
mutations that never had been reported [14]. This will provide new information for
69
researchers to study RDEB. Half of our patients showed the low level of circulating anti-
C7 antibodies, however, the circulating antibodies were not pathologic to patients since
we know by immunofluorescence staining that they do not bind to patients’ skin. A
dilemma always exists with C7 supplementary therapies because the autoimmunity could
cause RDEB patients to obtain EBA during C7 therapies. In our previous study of
intradermal injections of human rC7 on RDEB mouse model, the autoimmune responses
occurred, because the RDEB mouse model immune system had never encountered the
missing C7. Those RDEB mice developed circulating anti-C7 antibodies. Though they
were not pathological to mice since the anti-C7 antibodies did not deposit to the BMZ of
skin, even with abundant human rC7 at the mouse’s BMZ [15]. Similar results were
found in the pre-clinical RDEB patients’ selection. Although we did detect low-level of
circulating anti-C7 antibodies in 11 of 22 RDEB patients despite the varying C7
expression in BMZ, none of them deposited the autoantibodies to BMZ and caused EBA.
We did not find the relationships between having anti-C7 antibody and the type of
COL7A1 mutations, the patients’ age, or the type of RDEB. Recently, other studies
analyzed more RDEB patients, and some of them exhibited the anti-C7 antibodies.
However they did not disclose whether the autoantibodies were pathological to patients
as none of the studies performed direct immunofluorescence staining [16, 17].
Constant wounding and scarring accompany RDEB patients’ daily life, which
have not only heavily affected their normal life due to the fibrotic fusions of digits caused
by excessive scars, or nutritional deficiencies caused by narrow esophageal, fixed or
denudated tongue, microstomia and poor dentition, they also would have a cumulative
risk of developing aggressive squamous cell carcinoma of 70% by age 45, in comparison
to the regular rate at 40% around age 60 [7]. At the beginning of RDEB studies,
physicians and researchers believed the excessive scars are caused the depth of the RDEB
bullae. Nevertheless, the characterization of RDEB patients skin cells study suggested
that the absence of functional C7 leads to up-regulations of TGF β mediated fibrosis and
its downstream signaling, which could cause the extensive scarring. Although TGF β is
needed in the early stages of wound healing, the continued aberrant expression of TGF-
β1 causes the abnormal wound healing such as over production and deposition of the
ECM proteins on the wound bed. It has been reported that RDEB fibroblasts synthesis
70
more TGF-β1, tenascin C and collagen I, III, V and VI than normal human fibroblasts
[18]. In normal human tissue, TGF β is considered a tumor suppressor, and inhibits skin
keratinocytes proliferation. In tumorgenesis, however, they may contribute to cancer
progression with different doses while it depends on the types of tumor [19, 20].
Our previous animal studies conducted in normal mouse wound healing model,
with the topical human rC7 treatment, we observed an increase of anti-fibrotic TGF-β3,
and a decrease in fibrosis markers CTGF and α-SMA in rC7 treated wounds compared
with vehicle alone treated control wounds. In the RDEB patients’ samples, we utilized
immunofluorescence staining on patients’ skin and immunoblotting analysis on patients’
cells, we observed the up-regulated TGF-β1 and 2, the TGF-β R-I, downstream signaling,
p-Smad2/3, and fibrosis markers α-SMA, Col I, and fibronectin [5]. Chen’s lab also
showed the high affinity between C7 and TGF β isoforms in vitro by binding assays, and
conducted the fibroblast-populated collagen lattice contraction assay to show the
aggravated contraction activity in RDEB patient fibroblasts compared to normal
fibroblasts in the presence of growth factor at 16 hours. With those in vitro and in vivo
results indicating the relationships between C7 and TGF β mediated fibrosis pathways,
we proposed that the mechanism that excessive scarring in RDEB patients could be
caused by lack of inhibition of pro-fibrogenic TGF β isoforms due to the absent C7 in
RDEB patients. From the results of immunofluorescence staining on patients’ skin with
other TGF β non-conical pathway markers, we did not observe the up-regulations in Erk1
and p38, so MAP kinase pathway was excluded in the further experiments [21].
We demonstrated that the loss of C7 is directly responsible for the up-regulation
of fibrogenic TGF-β1 and the activation of TGF-β1-mediated fibrosis signal transduction
pathway. With lentiviral vectors we re-introduced C7 to the RDEB patients fibroblasts,
we observed the down-regulations of TGF-β1, p-Smad 2/3, α-SMA, C1 and Fibronectin
[3, 22]. With 20ug/ml C7 protein we directly added to RDEB fibroblasts cultures, the
results showed reduced TGF-β1 and α-SMA. We determined the dose of added C7 based
on the previous in vivo work, such as the optimized dose of topical rC7, to enhance the
wound healing and reduce the scar formation [5]. The stability and duration of C7 in the
fibroblasts medium were not determined, and the optimized time and dose courses were
71
not yet conducted. After the encouraging results we got from reintroducing C7 to RDEB
patients, we also went ahead to silent C7 expression in normal fibroblasts with siRNA
specific to C7 to see if we can reproduce the similar up-regulation of pro-fibrogenic TGF-
β and fibrosis markers. We mixed four published siRNAs, transfected into two different
normal fibroblasts, and observed the up-regulated TGF-β1, downstreams p-Smad2/3, and
fibrosis markers α-SMA, C1 and fibronectin by immunoblotting. The C7 knockdown in
fibroblasts is more complete than in keratinocytes [10]. Up to this point, Chen’s lab is
first to verify that the altered C7 expression caused over-activated TGF-β1, its
downstream signaling pathway, and the excessive ECM productions and deposition. We
provide the first evidence that over-activated TGF β pathways could be the reason for
RDEB patients having excessive scarring.
TGF β isoforms could induce the fibrosis through canonical pathway mediated by
the activated Smad transcription factors. In canonical signaling, TGF-β isoforms bind to
its TGF-βRII to initiate the signal transduction. The TGF-β R-I then is recruited and
phosphorylated to the complexity of TGF-β and TGF-β R-II. Active TGF-β R-I recruits
and phosphorylates downstream Smad2 and Smad3, allows its association with Smad4
forming into a complex that relocates into the nucleus and regulates the TGF-β target
genes in transcriptional levels. Figure 4.9 shows our current working model on how C7
regulates TGF β pathway and inhibits fibrosis and scarring. There is less or no C7 in
RDEB patients to bind the endogenous TGF-β1. Excess TGF-β1 binds to TGF-β R-II to
initiate the canonical pathway that causes a number of fibrosis markers to be over
produced and deposited to healed wound, such as α-SMA, Col I, fibronectin and tenascin.
As the consequences, the extra scars form. With the supplements of C7 in various
delivery methods, C7 could bind with excessive TGF-β1, and inhibit the downstream
activation, down-regulate the transcription of TGF β targeted genes, and reduce the ECM
protein productions and less scar formations [23]. To test the hypothesis on how RDEB
patients have excessive scar and fibrosis, we conducted a series of experiments along the
pathways. First, we added the pan-neutralizing antibody to TGF-β1 to RDEB fibroblasts
cultures and showed that it neutralized the TGF-β1 and reduced the α-SMA productions.
Second, we utilized a chemical inhibitor, SB505124, to inhibit the binding of TGF β R-I
to TGF β isoforms and found the down-regulation of canonical pathway p-Smad 2/3, and
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fibrosis markers, α-SMA and C1. Last, we tried to disconnect the canonical Smad-
dependent pathway in RDEB fibroblasts by knockdown the expression of Smad2 and 3
with siRNAs to Smad 2 and Smad 3 and observed the down-regulation of C1, fibronectin
and α-SMA. With three different approaches along the TGF β pathways, we
demonstrated that canonical TGF β pathway is highly involved in excessive scars in
RDEB patients. Moreover, the ability of C7 to bind to TGF-β1 may provide molecular
basis for its ability to prevent the excessive TGF-β1 induced extra ECM productions.
Among all three TGF β isoforms, both TGF-β1 and 2 are pro-fibrotic factors.
However, we kept our focus on the TGF-β1 signaling in the later research of this chapter,
because we did not detect the up-regulation of TGF-β2 in the conditioned medium of
RDEB patient fibroblasts. It does not mean that TGF-β2 is not involved in the RDEB
scarring and fibrosis. As seen in Figure 4.1a, TGF-β2 clearly has dramatic up-
regulations in the patients’ epidermis. Moreover, the other fibrosis indicators, such as
TGF β R-I and p-Smad 2/3, revealed more significant up-regulations in epidermis or
keratinocyte by immunofluorescence staining or immunostaining, respectively. Perhaps
TGF-β2 over-expresses in keratinocytes, and diffuses into the dermis to contribute to the
same signaling pathways, and synergies with other pro-fibrotic factors to contribute to the
RDEB scarring formation. RDEB keratinocytes may not directly cause the excessive
scarring, but act as a compliance since it could be the “powerhouse” of fibrosis factors
that stimulate the over expression of ECM proteins within the dermal fibroblasts [24, 25].
In summary, the screening of the RDEB patients for our ongoing clinical trial
with intradermal injection of human rC7 provided us a unique opportunity to
communicate, analyze, evaluate and do research with twenty-two RDEB patients. We
witnessed that the expression level of C7 are highly related to the severity of the disease
and the status of AFs. However, during the screening, we also met two pairs of siblings,
which had the same gene mutations but exhibited different disease severity. A paper
published a few months ago discussed this issue, and authors believed that the abnormal
TGF β signaling pathway may also play an important role in determining the disease
severity [26]. In this chapter, we used multiple approaches to demonstrate that the up-
regulated TGF β pro-fibrotic isoforms and their fibrosis pathways are involved in RDEB
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scarring and fibrosis. This is caused by the lacking of C7 in RDEB patients to counteract
the extra endogenous TGF β isoforms-mediated fibrosis signaling. Importantly,
understanding the mechanism behind the RDEB scarring and fibrosis could be useful for
designing molecular therapies to improve the life quality for RDEB patients, and reduce
the risk for RDEB patients from developing aggressive skin squamous cell carcinoma
that frequently take the patients’ life.
4.4 Methods and Materials
RDEB patient’s skin and blood samples collection
RDEB patients’ skin and blood samples were obtained from voluntary patients
who underwent a clinical trial phase I/II selection at Hospital of University of Southern
California. Sample collections were obtained after patients have agreed to give their skin
for research purposes, have read and signed the informed consent forms. This study was
approved by Research Ethical Committee University of Southern California (Human).
The skin biopsy was shaved by size 10 scalpel, the length-width-thickness are
approximately 6mm x 3mm x 1mm. Transferred to sterile PBS immediately, started
preparing samples accordingly within one hour after the biopsies. 5ml whole blood
samples were obtained from each patient, and only sera were applied in experiments.
Primary RDEB skin keratinocyte and fibroblasts isolation
Primary normal human skin keratinocytes and fibroblasts isolations methods are
as described. Briefly, an approximately size 3mm x 3mm x 1mm of RDEB patients’ skin
left in a P100 dish containing 10ml PBS with 25 caseinolytic units/ml dispase (BD
Biosciences, US) for 12 hours at 4 degree. Separated the epidermis and dermis carefully
by sterile forceps.
Epidermis is placed in trypsin-EDTA (0.25%, USC core facility, US) solution for
15min at 37 degree, and then neutralized by equal volume of 2x soybean trypsin inhibitor
(Cascade Biologics, US). The whole solutions were spinned down everything for 10min
at 3000rpm to obtain keratinocytes pellet, and suspended with PBS to wash the pellet.
74
After spinning down, suspended with keratinocyte growth medium (Epilife, Life
Technologies, US) and place them in a P100 plate that pre-coated with 29ug/ml Col I.
Dermis is minced into tiny pieces by sterile scissors, and left with 100 unit/ml
collagenase for 2-3 hours at 37 degree. Used the equal volume of 10% FBS containing
Dulbecco's Modified Eagle Medium Mixture with F-12 (DMEM/F12) (GIBCO, Life
Technology, US) to collect and neutralize the solution. The whole solutions were spun
down everything for 10min at 3000rpm to obtain fibroblasts pellet, and suspended with
PBS to wash the pellet. Spun down and suspended with 20% FBS containing
DMEM/F12 (GIBCO, Life Technology, US) medium and place them in a P100 plate,
change into 10% FBS DMEM/F12 medium 48h later.
Immunofluorescence staining and H&E of tissue
Five-micrometer thick sections of OCT-embedded frozen tissues were cut on a
cryostat, fixed for 5 minutes in cold acetone, and air-dried. Immunolabeling of the tissue
was performed using standard immunofluorescence methods as described previously.
Briefly, for single-sand double-immunofluorescence staining, sections were blocked with
normal goat serum (1:10, sigma) for 1 hour at room temperature. Primary antibodies
were diluted in phosphate buffered saline with 1% bovine serum albumin. For C7
staining, we used monoclonal antibodies against human C7, clone LH 7.2 (Sigma), or a
rabbit poly- clonal antibody that recognizes both mouse and human C7. For double-
immunofluorescence staining, we incubated the mouse monoclonal anti-human C7
antibody together with a rabbit polyclonal antibody to both human and mouse C7. For
TGF-staining, we used polyclonal antibodies against TGF- 1 (sc-146, Santa Cruz
Biotechnology, Santa Cruz, CA), TGF- 2 (sc-90, Santa Cruz Biotechnology). Other
used antibodies are anti-phosphorylated Smad 2/3 (#8828, Cell Signaling), anti-Smad 2
(Cell Signaling), anti-Smad 3 (Cell Signaling), anti-phosphrylated Smad 3 (#9520, Cell
Signaling, US), anti-CTGF (ab6992, Abcam), anti- -Actin (a5441, sigma), anti-
Fibronectin (F3648, Sigma), anti-p38 MAPK thr180/tyr182 (#4511, Cell Signaling), anti-
type I collagen (sc-8784, Santa Cruz), anti-SMA (A2547, Sigma), Anti Human TGF R-
I/ALK-5 (AF3025, R&D systems). All primary antibody dilutions were 1:200. After
75
incubation for 1 hour at room temperature, sections were washed in phosphate buffered
saline three times and stained for 1 hour with FITC-conjugated goat anti-mouse IgG1
with or without Cy3-conjugated goat anti-rabbit IgG with or without anti-goat IgG
(Sigma) diluted 1:500 in phosphate buffered saline with 1% bovine serum albumin.
Slides were mounted with 40% glycerol. H&E staining was carried out as previously
described. Photographs of stained sections were taken using a Zeiss Axioplan
fluorescence microscope equipped with a Zeiss Axiocam MRM digital camera system
(Carl Zeiss International, Göttingen, Germany).
siRNA transfections
For Col VII knockdown, RDEB patients fibroblasts cell lines were transfected
with a Mission siRNA of four synthetic siRNAs (Sigmaaldrich, US), targeting Col VII.
Transfection was performed according to the manufacturer's protocol and optimised for a
six-well plate. Briefly, cells were plated at 50% confluency and subjected to transfection
the following day using 4 μg of DharmaFECT1 (Thermo Scientific, US) transfection
reagent and 12.5 nM final concentration of each siRNA (apart from the assays where the
siRNA concentration was titrated). Transfection media were replaced with complete
DMEM/F12 media after 16 hours. ColVII protein expression was analysed by WB on
cell extract, 2-4 days post-transfection. Cells incubated with the transfection reagent,
Mission siRNA Universal Negative Control #1 only (Sigma aldrich, US) were used as
negative control. The conditions are similar for knockdown of Smad 2 and Smad 3,
except the final concentration was 160nM for Smad 2, Smad 3 and negative siRNA. The
sequence 5’-3’ of siRNAs are as follow,
C7 4-1: GGGCUUGGAUGGUGACAAAUU
C7 4-2: CAUCGCAUCUGGAUCACGAUU
C7 4-3: CCGCUGACAUUGUGUUCUUUU
C7 4-4: GAAUGUGGAUCGGUUGCUGUU
SMAD 2-1: CAACUCUUCUGUCAUAGCA
SMAD 2-2: CAUGUUAUAUAUUGCCGAU
SMAD 3-1: CCUUUGCAGUGGCUUGACA
SMAD 3-2: CAGUUCUACCUCCUGUGUU
76
Immunoblotting
To determine the cellular expression of TGF β pathway in RDEB patients’
keratinocytes and fibroblasts, cellular extracts were prepared 48 hours after subcultured
and subjected to 4-15% SDS-PAGE (Bio-Rad, Hercules, CA). Proteins were then
electrotransferred onto a nitrocellulose membrane. The presence of C7 was detected with
polyclonal antibodies to the NC1 domain of C7, followed by a horseradish peroxidase-
conjugated goat anti-rabbit IgG and enhanced chemiluminescence detection reagent (GE
Healthcare, UK). To detect protein expression in response to 5ng/ml TGF-β1 or 5ng/ml
TGF-β1 with 20ng/ml C7, cells were plated at a density of 2.5 × 10
5
cells/6 well plate,
allowed to attach for 24h, and starved with serum-free DMEM/F12 for overnight. Cells
were then treated with 5ng/ml TGF-β1 or 5ng/ml TGF-β1 with 20ng/ml C7 for the
indicated times (15 min–48 h). To detect protein expression and modification in response
to treatment with SB505124 or TGF β pan specific antibody, cells were plated at a
density of 2.5 × 10
5
cells/6 well plate and were cultured for the 48h in the presence or
absence of the inhibitor or antibody. Total protein was extracted with lysis buffer.
Lysates were then centrifuged at 12,000× g for 10 min at 4 °C. Protein concentrations
were determined using the Bradford assay kit (Bio-Rad, USA). Proteins were separated
using precast 4%–12% gradient SDS-PAGE (Invitrogen) and were transferred onto
0.45um nitrocellulose blotting membranes (GE Healthcare Life science, USA). Blots
were incubated with the indicated primary antibodies at 4 °C and horseradish peroxidase-
conjugated anti-mouse and anti-rabbit secondary antibodies (1:5000 dilution) at RT. The
primary antibodies anti-TGF β1, anti-TGF β2 and anti α-SMA dilution are 1:2000,
primary antibodies anti-TGF β R-I, anti-TGF β R-II dilution are 1:1000 and β-Actin
dilution is 1:5000. Blots were then visualized using a chemiluminescence detection
system according to the manufacturer’s instructions (GE Healthcare Life science, USA).
TGF-β1 and 2 concentrations by ELISA kits
RDEB patients’ fibroblasts were split and seeded 2.5x10
5
/well in a six-well plate,
change into 1.5 ml serum free DMEM/F12 and incubated at 37°C for 48h. Harvested the
77
medium and spun down to eliminate the debris. Quantification of TGF-β1 and TGF-β2
in RDEB patients’ fibroblasts was performed in triplicate by ELISA kit (DB100B and
DB250, R&D systems, US) according to the manufacturer’s instructions. The
concentrations of TGFβ1 and TGF-β2 in fibroblasts with different experiments were
analyzed with the kit. The optimal density of the color reaction was detected at a
wavelength of 450 nm and 605nm using a chemiluminescence reader. The background
signal detected at 450 nm and 605nm were subtracted from the determined values. Delta
values were normalized to the extinction obtained from standard curves, and protein
contents were calculated for each sample. TGF β isoform amounts were normalized to
identical numbers of cells per milliliter of medium.
Inhibition and Neutralization of TGF β signaling
RDEB patients’ fibroblasts were split and seeded 2.5x10
5
/well in a six-well plate,
which is 60-70% conflurency in the next day. The cells were incubated in serum
containing DMEM/F12 overnight, then treated 1.5ml serum free medium with TGF β
receptor I inhibitor, SB505124 or TGF β pan specific antibody at 37°C for 48h. Harvest
and subjected protein exactions to immunoblotting.
Immunofluorescence Staining of Cell Cultures
To determine the efficiency of lentiviral vector coding for full length Col VII
transduction, we subjected normal human fibroblasts or transduced RDEB patients’
fibroblasts were plated in TissueTek chamber slides (Nunc, Inc., Naperville, IL) on
polylysine at 37°C for 18 h, 4x10
5
cells per well. Cells were immersed in periodate-
lysine-paraformaldehyde fixative for 10 min at room temperature, washed several times
with PBS to remove fixative, and then permeabilized and blocked by incubating in PBS
with 3% BSA, 1% saponin, and 10% normal goat serum for 15 min at room temperature.
The cells were incubated with an affinity-purified polyclonal antibody to the NC1 domain
of type VII collagen at a dilution of 1:200 in a humidified chamber for 2 h, then washed
three times with PBS, 1% saponin, counterstained with a fluoresce in isothiocyanate-
conjugated goat antibody to rabbit IgG (1:200 dilution) for 1 h (Organon Teknika-
Cappel) and washed. The cells were then examined and photographed with a Zeiss
78
epiluminating immunofluorescence microscopy.
Cell Cultures
The human primary dermal fibroblasts cell line was cultured in Dulbecco's
modified essential medium (DMEM) supplemented with 20 % fetal bovine serum (FBS)
after isolation. After split confluent cultures, used (DMEM)/Ham’s F12 (1:1)
supplemented with 10% FBS. The human primary epidermal keratinocytes cell line was
cultured in Epilife keratinocyte medium supplemented with Human Keratinocyte Growth
Supplement, including bovine pituitary extract (BPE), 0.2% v/v; recombinant human
insulin-like growth factor-I, 1 µg/ml; Hydrocortisone, 0.18 µg/ml; bovine transferrin, 5
µg/ml; human epidermal growth factor, 0.2 ng/ml. Primary fibroblasts and keratinocytes
were passaged as they reached confluence. Fibroblasts all experiments were performed
on cells between passages 4 to 12, keratinocytes all experiments were performed on cells
between passages 3 to 6.
79
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collagen homes to skin wounds and restores skin integrity of dystrophic
epidermolysis bullosa. J Invest Dermatol, 2013. 133(7): p. 1910-1913.
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incorporates into the dermal-epidermal junction and promotes wound closure.
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dystrophic epidermolysis bullosa. Nat Genet, 2002. 32(4): p. 670-5.
10. Martins, V.L., et al., Increased invasive behaviour in cutaneous squamous cell
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122(Pt 11): p. 1788-99.
11. Ghosh, A.K., S.E. Quaggin, and D.E. Vaughan, Molecular basis of organ
fibrosis: potential therapeutic approaches. Exp Biol Med (Maywood), 2013.
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12. Zhao, J., et al., Smad3 deficiency attenuates bleomycin-induced pulmonary
fibrosis in mice. Am J Physiol Lung Cell Mol Physiol, 2002. 282(3): p. 585-593.
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14. Woodley, D.T., et al., De novo anti-type VII collagen antibodies in patients with
recessive dystrophic epidermolysis bullosa, in J Invest Dermatol. 2014: United
States. p. 1138-40.
15. Remington, J., et al., Injection of recombinant human type VII collagen corrects
the disease phenotype in a murine model of dystrophic epidermolysis bullosa. Mol
Ther, 2009. 17(1): p. 26-33.
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16. Pendaries, V., et al., Immune reactivity to type VII collagen: implications for gene
therapy of recessive dystrophic epidermolysis bullosa. Gene Ther, 2010.
17. Tampoia, M., et al., Prevalence of specific anti-skin autoantibodies in a cohort of
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18. Kuttner, V., et al., Global remodelling of cellular microenvironment due to loss of
collagen VII. Mol Syst Biol, 2013. 9: p. 657.
19. Stanley, J.R., N. Rubinstein, and V. Klaus-Kovtun, Epidermolysis bullosa
acquisita antigen is synthesized by both human keratinocytes and human dermal
fibroblasts. J Invest Dermatol, 1985. 85(6): p. 542-545.
20. Chen, M., et al., The carboxyl terminus of type VII collagen mediates antiparallel
dimer formation and constitutes a new antigenic epitope for epidermolysis
Bullosa acquisita autoantibodies. J Biol Chem, 2001. 276(24): p. 21649-21655.
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23. Wynn, T.A., Cellular and molecular mechanisms of fibrosis. J Pathol, 2008.
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81
Figures:
Table 4.1 Summary of C7 expression and AFs in RDEB patients’ skin and anti-C7 antibodies in the blood
Patient
ID
Patient
Age
C7 Expression at
DEJ
AF by Electron Microscoy NC1/NC2
ELISA
C7
ELISA
C7 Western
Blot
Clinical
Diagnosis
Density Morphology
1 24 Reduced + Very thin and wispy ± + + RDEB-sev,gen
2 6 Absent 0 Absent − − − RDEB-sev,gen
3 10 Absent 0 Absent − − − RDEB-sev,gen
4 27 Reduced 0 Absent − − − RDEB-sev,gen
5 25 Reduced ++ Thin, rarely arching + + + RDEB-sev,gen
6 24 Reduced +++ Thin, rarely arching + + + RDEB-sev,gen
7 36 Reduced ++ Thin, rarely banded, rarely arching − − − RDEB-sev,gen
8 11 Absent + Short, rudimentary + + + RDEB-sev,gen
9 5 Reduced ++ Straight, non-banded + + + RDEB-sev,gen
10 3 Reduced + Thin, mild arching − − − RDEB-sev,gen
11 34 Absent + Short, rudimentary − − − RDEB-sev,gen
12 27 Reduced + Thin and wispy − + + RDEB-sev,gen
13 22 Reduced + Thin and wispy − − − RDEB-sev,gen
14 37 Normal ++++ Few banded, arching, looped − − − RDEB-I
15 23 Normal +++ Non-banded, arching − − − RDEB-I
16 28 Normal +++++ Banded, arching − + + RDEB-I
17 62 Normal ++++ Thin, arching, looped − − − RDEB-I
18 11 Normal +++ Non-banded, some arching + − + RDEB-I
19 38 Normal ++++ Banded, arching − + + RDEB-I
20 4 Normal + Very thin and straight + + + RDEB-O
21 45 Normal ++++ Thin and wispy, occasionally mild arching + + + RDEB-O
22 31 Normal +++ Thin, wispy, occasional arching − + + RDEB-O
NHS Normal +++++ Thick, banded, arching, looping − − −
EBA − − − + + +
82
Figure 4.1 The profiling of TGF β pathway markers in RDEB patients. (a)
Immunofluorescence staining of normal human and RDEB patients’ skin were performed
with antibodies specific for TGF β pathway markers, TGF β-1, TGF β-2 and p-Smad 2/3,
and fibrosis markers, Col I and FIN. The patients’ skin revealed the up-regulation of all
markers in RDEB patients’ skin compared to normal human skin. (b) The ratio
differences between NHS and RDEB patient’s skin as evaluated by Image J analysis.
83
Figure 4.1 The profiling of TGF β pathway markers in RDEB patients. (c)
Immunoblotting of RDEB keratinocytes with TGF β pathway markers. Cell lysates were
prepared and then subjected to 4-12% SDS-PAGE, followed by immunoblotting analysis
with specific antibodies to TGF β-1, TGF β-2, TGF β R-I, p-Smad 2/3 and anti β-Actin
antibody. All markers on RDEB keratinocytes displayed up-regulations compared to
normal human keratinocytes. (d) The chart shows the ratio differences in all markers
between RDEB keratinocytes and normal human keratinocytes. (e) Immunoblotting of
RDEB fibroblasts with TGF β pathway markers. Cell lysates were prepared and then
subjected to 4-12% SDS-PAGE, followed by immunoblotting analysis with specific
antibodies to TGF β pathway markers, p-Smad 2/3, TGF β R-I and TGF β R-II, fibrosis
markers α-SMA and Col I, and to internal control β-Actin. All markers on RDEB
fibroblasts showed up-regulations when compared to normal human fibroblasts. (f) The
chart shows the ratio differences in all markers between RDEB fibroblasts and normal
human fibroblasts.
84
Figure 4.1 The profiling of TGF β pathway markers in RDEB patients. (g) Levels of
TGF-β1 in conditioned media of RDEB fibroblasts and normal human fibroblasts 48
hours after culturing as assessed by ELISA. P values<0.05 after adjustment for multiple
comparisons are indicated. (f) Levels of TGF-β1 in RDEB patients’ sera also showed
elevated numbers when compared to normal human sera by ELISA. P values<0.05 after
adjustment for multiple comparisons are indicated.
85
Figure 4.2 Lentiviral vector mediated gene transfer of C7 to RDEB fibroblasts
reduces the expressions of TGF β pathway and fibrosis markers. (a) Immunoblot
analysis of lentiviral vector transduced RDEB fibroblasts and the same patient non-
transduced fibroblasts. Cell lysates were prepared and then subjected to 4-12% SDS-
PAGE, followed by immunoblot analysis with specific antibodies to C7, p-Smad 2/3 and
fibrosis markers α-SMA, Col I and FIN. β-Actin was included as a loading control. (b)
The chart shows the ratio differences between non-transduced and lentiviral transduced
RDEB fibroblasts. They are based on the strength of signals after adjusted by the loading
of β-Actin. (c) Immunofluorescence staining of RDEB transduced fibroblasts with an
affinity-purified polyclonal antibody to the NC1 domain of C7. We achieved a high
efficiency of C7 gene transfer to RDEB fibroblasts. (d) Levels of TGF-β1 in 48 hours
incubated fibroblast conditioned media from non-transduced and transduced RDEB
fibroblasts by ELISA. Normal human fibroblasts serve as negative control. P values<0.05
after adjustment for multiple comparisons are indicated.
86
Figure 4.3 Lentiviral vector mediated gene transfer of C7 to RDEB keratinocytes
reduces the expression of TGF β pathway markers. (a) Immunoblot analysis of
lentiviral vector transduced RDEB keratinocytes and the same patient non-transduced
keratinocytes. Cell lysates were prepared and then subjected to 4-12% SDS-PAGE,
followed by immunoblot analysis with specific antibodies to C7, TGF-β1, TGF-β2 and p-
Smad 2/3. β -Actin was included as a loading control. (b) The chart shows the ratio
differences between non-transduced and lentiviral transduced RDEB keratinocytes. They
are based on the strength of signals adjusted by the loading of β-Actin. (c)
Immunofluorescence staining of RDEB transduced keratinocytes with an affinity-purified
polyclonal antibody to the NC1 domain of C7. We achieved 43.8% transduction
efficiency of C7 gene transfer to RDEB keratinocytes. NKC, normal human
keratinocytes.
87
Figure 4.4 Supplementing C7 or NC1 to RDEB fibroblasts decreases the expression
of TGF β1 induced the activation of p-Smad 2/3. (a) RDEB fibroblasts were serum
starved for 18 hours and then stimulated with TGF-β1 (5ng/ml) in the presence or
absence of C7 or NC1 for 15 minutes. Cell lysates were prepared and subjected to 4-12%
SDS-PAGE followed by immunoblot analysis with antibody specific to p-Smad2/3. Panel
a reveals that C7 or NC1 inhibits TGF-β1 induced p-Smad 2/3 activation in RDEB
fibroblasts. β-Actin was included as a loading control. (b) The chart shows the ratio
differences between non-transduced and lentiviral transduced RDEB fibroblasts. They are
based on the strength of signals adjusted by the loading of β-Actin. (c) Levels of TGF-β1
in 48 hours incubated fibroblasts culture supernatants from C7 treated and untreated
RDEB fibroblasts by ELISA. P values<0.05 after adjustment for multiple comparisons
are indicated.
88
Figure 4.5 siRNA mediated knockdown of C7 in normal fibroblasts increases the
expression of TGF-β pathway and fibrosis markers. (a) Immunoblot analysis of
siRNA knockdown C7 in normal human fibroblasts, and random scrabbled siRNA as
experimental control. Cell lysates were prepared and then subjected to 4-12% SDS-
PAGE, followed by immunoblotting analysis with specific antibodies to C7, Col I, and α-
SMA. β-Actin was included as a loading control. (b) Panel b is computer-generated
ratio differences between random scrabbled siRNA and C7 specific siRNA knockdown
normal human fibroblasts. They are based on the strength of signals the adjusted by the
loading of β-Actin. Error bars represent standard deviation of three different
experiments.
Figure 4.6 TGF-β pan specific
antibody reduces the expression
of TGF-β1 and α-SMA in RDEB
fibroblasts. (a) RDEB fibroblasts
treated with TGF-β pan specific
antibody for 48h. Cell lysates were
prepared and subjected to 4-12%
SDS-PAGE followed by
immunoblot analysis with antibody
specific to α-SMA. β-Actin was
included as a loading control. (b)
The chart shows the ratio difference
of levels of α-SMA between TGF-β pan specific antibody treated and untreated RDEB
89
fibroblasts. (c) Levels of TGF-β1 in 48 hours incubated fibroblasts culture supernatants
from C7 treated and untreated RDEB fibroblasts by ELISA. P values<0.05 after
adjustment for multiple comparisons are indicated.
Figure 4.7 TGF-β R-I inhibitor decreases the
expression of α-SMA in RDEB fibroblasts. (a) RDEB
fibroblasts treated with TGF-β R-I inhibitor SB505124 for
48h. Cell lysates were prepared and subjected to 4-12%
SDS-PAGE followed by immunoblot analysis with
antibody specific to α-SMA. β-Actin was included as a
loading control. (b) The chart shows the ratio difference of
levels of α-SMA between TGF-β R-I inhibitor treated and
untreated RDEB fibroblasts.
Figure 4.8 siRNA mediated
knockdown of Smad-2 or
Smad-3 in RDEB fibroblasts
decreases the expression of
TGF-β pathway and fibrosis
markers. (a) Immunoblot
analysis of siRNA
knockdown Smad-2 or Smad-3
in RDEB human fibroblasts,
and random scrabbled siRNA
as experimental control. Cell
lysates were prepared and then
subjected to 4-12% SDS-
PAGE, followed by
immunoblot analysis with
specific antibodies to smad-2,
smad-3 and α-SMA. β-Actin
was included as a loading
control. Results show the
significance and specification
of both siRNA Smad-2 and
90
Smad-3 knockdown. (b) Panel b is computer-generated ratio differences between random
scrabbled siRNA and Smad-2 or Smad-3 specific siRNA knockdown in RDEB
fibroblasts. They are based on the strength of signals the adjusted by the loading of β-
Actin.
Figure 4.9 The schematic model of how C7 responsible for altered TGF-β fibrosis
signaling and hyper-contraction activity in RDEB fibroblasts. TGF-β induced
fibrosis is through Smad signaling mediated by the activation of Smad transcription
factors.
TGF-β acts by binding initially to TGF-β R-II, then TGF-β R-I is recruited to the
TGF-β-TGF-β R-II receptor complex and becomes phosphorylated. Active TGF-β R-I
phosphorylates Smad2 or Smad3, allowing its association with Smad4 into a complex
that relocates into the nucleus and mediates the transcriptional regulation of TGF-β target
genes. With the presence of C7 or down-regulated/abolished TGF β pathway, the
expressions of fibrosis markers, α-SMA, C1 and Fibronectin, would be reduced, thus, less
scars formed.
Abstract (if available)
Abstract
Dystrophic epidermolysis bullosa (DEB) is a family of rare inherited mechano-bullous disorders caused by mutations in the gene encoding for type VII collagen (C7), the major component of anchoring fibrils (AF). These mutations cause perturbations in AFs, structures that hold the outer epidermal layer of skin onto the inner dermal connective tissue layer. Without sufficient functioning AFs, the epidermis separates from the dermis. Children with DEB are born with skin fragility and continuous blistering of the skin in trauma-prone sites. Eventually, most of these children die from aggressive squamous cell carcinoma at the sites of chronic blistering and scarring. The C7 gene knockout mouse model recapitulates the clinical, genetic and ultrastructural features of human recessive DEB. ❧ We evaluated the feasibility of C7 based protein therapeutics via intradermal injection (ID) of recombinant human C7 (rC7) into DEB mice or topical application of rC7 onto skin wounds created onto normal mice or engrafted DEB mouse skin. With both administrations, we found that rC7 migrated and incorporated into the mouse’ basement membrane zone (BMZ), enhanced adherence of dermal epidermal junction, and reduced blistering formation. The most pivotal benefit of ID C7 is the prolonged life expectancy for DEB mice. Based on the preclinical studies, we conclude that protein based therapeutics may be feasible for the treatment of human patients with DEB. ❧ Moreover, the wounds treated with topical rC7 exhibited accelerated wound closure and less scar formation. Topical rC7 decreased the expression of pro-fibrogenic TGF-β2 and increased the expression of anti-fibrogenic TGF-β3, accompanied by the reduced expression of CTGF, fewer α-SMA positive myofibroblasts, and less deposition of collagen in the healed neodermis, consistent with less scar formation. In addition, we obtained skin and serum samples from DEB patients, and discovered that DEB patients display increased expression of pro-fibrotic TGF-β isoforms as well as markers associated with fibrosis. With siRNA knockdown C7 in normal fibroblasts and re-introduction of C7 to DEB patients’ cells, we confirmed that C7 loss is directly responsible for the up-regulated TGF-β signaling associated with fibrosis. These studies provided the first evidence of the potential use of topical C7 for accelerating scar-less wound healing. These findings suggest that C7 based protein therapeutics could be used for the treatments of DEB patients as well as accelerate wound healing with less scar formation in patients with chronic skin wounds.
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Asset Metadata
Creator
Wang, Xinyi
(author)
Core Title
The roles of type VII collagen in wound healing and scar reduction
School
Keck School of Medicine
Degree
Doctor of Philosophy
Degree Program
Pathobiology
Publication Date
10/20/2014
Defense Date
08/28/2014
Publisher
University of Southern California
(original),
University of Southern California. Libraries
(digital)
Tag
C7,DEB,dystrophic epidermolysis bullosa,OAI-PMH Harvest,protein-based therapeutics,rare disease,scar reduction,TGF beta pathways,type VII collagen,wound healing
Format
application/pdf
(imt)
Language
English
Contributor
Electronically uploaded by the author
(provenance)
Advisor
Hofman, Florence M. (
committee chair
), Chen, Mei (
committee member
), Chuong, Cheng-Ming (
committee member
), Frenkel, Baruch (
committee member
)
Creator Email
xinyi.wang@usc.edu,xinyi82@gmail.com
Permanent Link (DOI)
https://doi.org/10.25549/usctheses-c3-506591
Unique identifier
UC11297626
Identifier
etd-WangXinyi-2934.pdf (filename),usctheses-c3-506591 (legacy record id)
Legacy Identifier
etd-WangXinyi-2934.pdf
Dmrecord
506591
Document Type
Dissertation
Format
application/pdf (imt)
Rights
Wang, Xinyi
Type
texts
Source
University of Southern California
(contributing entity),
University of Southern California Dissertations and Theses
(collection)
Access Conditions
The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...
Repository Name
University of Southern California Digital Library
Repository Location
USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA
Tags
C7
DEB
dystrophic epidermolysis bullosa
protein-based therapeutics
rare disease
scar reduction
TGF beta pathways
type VII collagen
wound healing