ed-a fibronectin: a storage site for latent tgf -1 in the by · ed-a fibronectin: a storage site...
TRANSCRIPT
ED-A Fibronectin: A Storage Site for Latent TGF -1 in the
Myofibroblast Matrix?
by
Grace Wei Ye Chau
A thesis submitted in conformity with the requirements
for the degree of Master’s of Science
Faculty of Dentistry
University of Toronto
© Copyright by Grace Wei Ye Chau 2012
ii
ED-A Fibronectin: A Storage Site for Latent TGF -1in the Myofibroblast Matrix?
Grace Chau
Master’s of Science
Faculty of Dentistry
University of Toronto
2012
Abstract
Fibrosis, a major cause of organ failure, has no effective therapy available. Responsible for
fibrosis are myofibroblasts. Mechanical stress, TGF -1 and ED-A FN are pivotal elements for
myofibroblast differentiation, however the exact link remains elusive. I hypothesize that ED-A
FN stores the latent TGF -1 in the ECM by interacting with the latent TGF -1 binding protein
(LTBP-1), and that matrix stiffness is a regulator of ED-A FN and LTBP-1.
Using co-IP and ED-A domain antagonists, ED-A FN and LTBP-1 associated in the ECM
of human dermal fibroblasts (HDFs). The effects of the 11th
_ED-A_12th
recombinant FN peptide
was most prominent in blocking LTBP-1 incorporation in the ECM. HDFs seeded on collagen-
coated substrates, showed an increase in expression and organization for both proteins with
matrix stiffness. In conclusion, the ED-A domain may require the aid of heparin linkages
flanking the 12th
domain of FN to bind to LTBP-1 in the ECM.
iii
Acknowledgements
I want to thank first my supervisor, Dr. Boris Hinz for providing me the guidance and
encouragement during my two years of studies. My two committee members: Dr. Michael
Glogauer and Dr. Craig Simmons, for their ideas and inspirations. Next, is my collaborator and
mentor, Dr. Eric White, who without his support, and belief in me, I would not be where I am
today. My other collaborators, Dr. Rebecca Wells, Dr. Tara Moriarty, and Dr. Ben Alman who
provided me with the materials I need to complete my project. Last but not least, my lab
associates (Jenna Balestrini, Anne Koehler, Melissa Chow, Charles Godbout, Franco Klingberg,
Yong Kwon, Vincent Sarrazy, Nilesh Talele, and Elena Zimina) and colleagues, who made my
life in the lab enjoyable and memorable.
iv
Table of Contents
Chapter 1: Introduction .................................................................................... 1
1.1 Hypothesis and Objectives ..............................................................................................1
1.2 Wound Healing: An Overview ........................................................................................2
1.3 The Inflammatory Phase .................................................................................................2
1.3.1 Inflammation: Vascular Response and Haemostasis ........................................................................ 5
1.3.2 Inflammation: The Cellular Response .............................................................................................. 6
1.4 Remodelling Phase: Formation of Granulation and Scar Tissue ....................................7
1.5 Fibroblast-to-Myofibroblast Differentiation in Tissue Repair ......................................10
1.5.1 Myofibroblast Features ................................................................................................................... 10
1.5.2 Origins of the Myofibroblast .......................................................................................................... 11
1.5.2.1 Resident Fibroblasts .............................................................................................................. 12
1.5.2.2 Circulating Precursors ........................................................................................................... 12
1.5.2.3 Epithelial-to-Mesenchymal Transition - EMT ...................................................................... 13
1.5.3 Mediators and Modulators of the Myofibroblast ............................................................................ 13
1.5.3.1 The Role of Growth Factors in Fibroblast Fibrogenesis........................................................ 13
1.5.3.2 Signal Transduction in Fibroblasts ........................................................................................ 14
1.6 The Myofibroblast ECM ...............................................................................................17
1.6.1 Fibronectins (FNs) .......................................................................................................................... 17
1.6.2 ED-A Fibronectin (ED-A FN) ........................................................................................................ 19
1.6.3 ED-A FN Expression ...................................................................................................................... 20
1.6.4 Generation of ED-A FN Occurs through Alternative Splicing ....................................................... 20
1.6.5 Integrin(s) Associated with ED-A FN............................................................................................. 21
1.6.6 ED-A FN as a Pro-Fibrotic Factor .................................................................................................. 22
1.6.6.1 Lung Fibrosis ......................................................................................................................... 24
1.6.6.2 Dupuytren’s Disease.............................................................................................................. 25
1.6.6.3 Atherosclerosis ...................................................................................................................... 25
1.6.7 The Role of ED-A FN in Myofibroblast Differentiation ................................................................ 26
1.7 The Link Between TGFβ-1 and the ECM .....................................................................27
1.7.1 The Activation of Latent TGFβ-1 ................................................................................................... 27
1.7.2 Latent Transforming Growth Factor Binding Proteins (LTBPs) .................................................... 29
1.7.3 Association of LTBP-1 with the ECM ............................................................................................ 30
1.8 The role of ECM Stiffness in Fibrogenesis ...................................................................31
Chapter 2: Materials and Methods ................................................................ 34
2.1 Preparation of Deformable Silicone Substrates .............................................................34
v
2.2 Culture and Analysis of Fibroblasts on Soft Substrates ................................................34
2.3 Recombinant FN Peptides and FN Constructs ..............................................................35
2.4 Solid Phase Binding Assay ............................................................................................36
2.5 ED-A Blocking with IST-9 Antibody and Recombinant Peptides ................................36
2.6 Co-Immunoprecipitation of LTBP-1 and ED-A FN .....................................................37
2.7 LTBP-1 Expression in Wild-type ED-A and ED-A-/-
Mice ..........................................37
Chapter 3: Results ............................................................................................ 39
3.1 LTBP-1 binds to ED-A FN in Vitro Mainly in the ECM of HDFs ...............................39
3.2 ECM Stiffness affects Co-Expression of ED-A FN and LTBP-1 .................................40
3.3 The 11th
_ED-A_12th
domain in FN appears to be a Binding Partner of LTBP-1 in the
Myofibroblast ECM ......................................................................................................43
3.4 LTBP-1 Expression is Lower in EDA-/-
Mice compared to Wild-type .........................48
Chapter 4: Discussion ...................................................................................... 50
4.1 Discussion .....................................................................................................................50
4.1.1 The role of ECM stiffness in LTBP-1 binding to ED-A FN ........................................................... 51
4.1.2 Specific binding of LTBP-1 to ED-A?............................................................................................ 53
4.1.3 Crosslinking of LTBP-1 with the ED-A FN ECM .......................................................................... 55
4.1.4 The role of ED-A FN binding integrins and alternative factors ...................................................... 56
4.2 Conclusion - ED-A FN as a Therapeutic Anti-Fibrosis Target? ...................................57
4.3 Outlook ..........................................................................................................................59
vi
List of Figures
Figure 1: The different stages of wound repair: a focus on the myofibroblast. .............................. 4
Figure 2: Origins of the myofibroblast. ........................................................................................ 12
Figure 3: TGFβ-1-induced-α-SMA transcription in myofibroblasts ............................................ 16
Figure 4: Cartoon schematic of one arm of FN ............................................................................ 18
Figure 5: Mechanisms of ED-A through Alternative Splicing. .................................................... 21
Figure 6: Model of myofibroblast contraction-mediated TGFβ-1 activation. .............................. 28
Figure 7: The Young’s Modulus of Tissues. ................................................................................ 32
Figure 8: Co-immunoprecipitation of LTBP-1 and ED-A FN...................................................... 39
Figure 9: The effect of ECM stiffness on the expression levels of ED-A FN and LTBP-1. ........ 41
Figure 10: The effect of stiffness on ED-A FN and LTBP-1 organization. ................................. 42
Figure 11: Effect of ECM stiffness and TGFβ-1 on ED-A FN and LTBP-1 organization. .......... 43
Figure 12: Purification of recombinant FN peptides. ................................................................... 44
Figure 13: Production and Purification of full FN constructs....................................................... 44
Figure 14: Blocking ED-A with IST-9 affects LTBP-1 incorporation into the ECM. ................. 45
Figure 15: The effect of recombinant peptides on LTBP-1 incorporation into the ECM............. 46
Figure 16: The effect of recombinant peptides on LTBP-1 incorporation into the ECM............. 47
Figure 17: The effect of recombinant peptides on LTBP-1 secretion into the medium. .............. 48
Figure 18: LTBP-1 Expression in wild-type and EDA-/- mouse fibroblasts................................ 49
Figure 19: Schematic of the PI3K/Akt/mTOR axis involved in the regulation of ED-A FN
alternative splicing and fibroblast activity. ................................................................................... 56
Figure 20: Potential therapeutic strategies targeting the differentiation of myofibroblast. .......... 59
vii
Abbreviations
-SMA = Alpha smooth muscle actin
ECM = Extracellular matrix
EMT = Epithelial mesenchymal transition
FAK = Focal adhesion kinase
FN= Fibronectin
bFGF = Basic fibroblast growth factor
CTGF= Connective tissue growth factor
cFN = Cellular fibronectin
IPF = Idiopathic pulmonary fibroblasts
LAP = Latency associated protein
LTBP-1 = Latent TGF -1 binding protein 1
MSC = Mesenchymal stem cells
TGF -1 = Transforming growth factor -1
PDGF = Platelet derived growth factor
PMN = Polymorphonuclear
VEGF = Vascular endothelial growth factor
MMP = Matrix metalloproteinase
MCP-1 = Monocyte chemoattractant protein-1
TIMP = Tissue inhibitor of metalloproteinases
1
Chapter 1: Introduction
1.1 Hypothesis and Objectives
Fibrosis is a major cause of organ failure with no effective therapy available.
Responsible for the detrimental conditions of fibrosis are myofibroblasts. Three factors
are pivotal for myofibroblast differentiation and function: active transforming growth
factorβ-1 (TGFβ-1), a mechanically resistant extracellular matrix (ECM), and the cellular
fibronectin (FN) splice variant ED-A FN (Hinz, 2009). TGFβ-1 is the most potent pro-
fibrotic cytokine known; it causes excessive ECM production, induces its own secretion
and drives myofibroblast differentiation (Grainger, 2007; Hinz, 2007; Leask and
Abraham, 2004; Ruiz-Ortega et al., 2007). Myofibroblasts secrete TGFβ1 together with a
latency-associated peptide (LAP). Association of this small latent complex with the latent
TGFβ-1 binding protein-1 (LTBP-1) provides a reservoir of latent TGFβ1 in the ECM by
binding to FN. Myofibroblasts pull on the large latent complex (LLC) to activate TGFβ-
1; this process requires anchorage of latent TGFβ-1 to stiff ECM (Wipff et al., 2007).
In my thesis, I investigate the role of the myofibroblast-characteristic FN splice
variant ED-A FN in the ECM anchoring process. ED-A FN is a pivotal element in
myofibroblast differentiation, but the mechanism of its action is virtually unknown. ED-
A FN is neo-expressed in healing wounds and up-regulated in organ fibrosis (Muro et al.,
2008; Serini and Gabbiani, 1996). ED-A FN deficient mice are protected from
experimentally induced lung fibrosis, and exhibit significantly reduced numbers of
myofibroblasts. ED-A-null mice fail to develop lung fibrosis and present a diminished
capacity for activation of TGFβ1 (Muro et al., 2008). I hypothesize that ED-A FN
exhibits specific characteristics in interacting with LTBP-1. FN is a major binding partner
of LTBP-1 in the ECM of fibroblasts (Unsold et al., 2001). However, neither the FN
splice variant specificity nor the influence of stress have been assessed in this interaction.
2
Hypothesis:
ED-A FN stores latent TGFβ-1 in the ECM by interacting with latency LTBP-1. The
expression levels of ED-A FN and LTBP-1 are co-regulated by ECM stiffness.
Objectives:
1. Determine whether LTBP-1 binds to ED-A FN.
2. Determine if ECM stiffness co-regulates the expression of ED-A FN and LTBP-1.
1.2 Wound Healing: An Overview
Tissue remodelling and closure of open wounds or tissue defects are two essential
processes that occur during normal wound healing (Tomasek et al., 2002). Although as
simple as “healing” may sound, it is in fact an intricate, complex and dynamic process
involving the interplay among local tissue cells, cells that are recruited to sites of injury,
and other components of the ECM. The complex process of wound healing can be
subdivided into three main phases: inflammation, tissue regeneration and subsequent
tissue remodelling. In the latter phase, so-called myofibroblasts are known to play a dual
role as the “good” for proper wound closure and repair, and the “bad” in promoting
fibrosis when they are deregulated (Fig. 1). Myofibroblasts are highly contractile cells
that are characterized by neo-expression of α-smooth muscle actin ( -SMA)which is
incorporated into stress fibres. Although the principal processes leading to tissue repair
are common to all injured tissues, I will here use skin wound healing as an example since
in my project I am working with skin myofibroblasts.
1.3 The Inflammatory Phase
Tissue injury often begins with the damage of blood vessels and leakage of blood
constituents into the extra-vascular space. Inflammation is the initial innate response that
occurs in the body after injury, and is a critical step needed for the subsequent tissue
repair and restoration processes. The inflammatory phase can be subdivided into four
stages, in which haemostasis is the first to occur. There are two types of inflammatory
response: vascular and cellular (Midwood et al., 2004). The cellular response occurs
3
when blood vessels erupt after tissue injury. When platelets arrive to the site of injury,
these cells adhere and aggregate to stop the bleeding forming a fibrin clot (Fig 1a). In
addition, platelets also are involved in releasing growth factors and other
chemoattractants to assist in the coagulation cascade (Singer and Clark, 1999). The
provisional ECM that the platelets form provides a temporary scaffold for recruiting cells
such as neutrophils to help remove foreign particles and remove damaged tissue. In
addition, other leukocytes such as macrophages help to release growth factors and other
cytokines, thereby initiating the formation of granulation tissue (Fig 1b) (Werner and
Grose, 2003).
4
Figure 1: The different stages of wound repair: a focus on the myofibroblast.
Figure 1: The different stages of wound repair: a focus on the myofibroblast.
In normal tissues, fibroblasts experience very low level of mechanical stress as they are shielded
from stress by their surrounding ECM. During tissue injury, an inflammatory reaction is
generated and the wounding site is a capped by a fibrin clot composed of platelets and fibrin.
The clot stops bleeding and prevents further entry of foreign antigens directly into the wound.
Growth factors and cytokines such as TGF -1 are secreted by local inflammatory and
immunogenic cells and act as chemoattractants to stimulate the migration of fibroblasts into the
wounding site. Simultaneously, angiogenesis begins to fill the wound, forming the granulation
tissue. At this stage, the ED-A FN splice variant is being produced by fibroblasts (a). As
fibroblasts migrate towards the open wound, they exert tractional forces on the collagen matrix,
generating mechanical stress in the ECM. In addition to this stress, the presence of both TGFβ-1
and increased levels of ED-A FN induce myofibroblast differentiation. Myofibroblasts are
highly contractile cells that are characterized by neo-expression of -SMA which is
incorporated into stress fibres (b). Differentiated myofibroblasts begin to deposit ECM
components such as collagen type I, and proteases to regulate tissue remodelling. The
continuous deposition of ECM proteins and contraction of the open wound is essential in the
proper tissue regeneration and wound closure (c). Closure of the wound signals apoptosis,
resulting in a formation of a scar (d). The persistence and deregulation of myofibroblast activity
leads to the pathological development of fibrosis such as hypertrophic scar (e) (Reproduced
from: (Tomasek et al., 2002)).
5
1.3.1 Inflammation: Vascular Response and Haemostasis
In the vascular response, the surrounding blood vessels become dilated, causing the
blood and plasma fluid to leak into the extra-vascular space, while preventing the lymph
from draining. This initiates the four cardinal signs of inflammation, starting with
redness, swelling, and later, heating, in which this entire process can last between one or
two days to as long as two weeks (Eming et al., 2007). The disruption of blood vessels
after tissue injury initiates haemostasis, which is a two- step process involving the
formation of a fibrin clot and subsequent coagulation (Verhamme and Hoylaerts, 2009).
Platelets are the first to infiltrate the area to maintain normal haemostasis. In this phase,
the adhesive platelets aggregate and subsequently release mediators such as serotonin,
adenosine diphosphate, and thromboxane A2, adhesive proteins like fibrinogen, FN,
thrombospondin, and von Willebrand factor VIII, that work together with the local
thrombin in forming the platelet plug or fibrin clot (Midwood et al., 2004).
In the second phase of haemostasis, coagulation involves an intrinsic and extrinsic
cascade. The intrinsic pathway is initiated by Hageman factor XII, a specific blood
enzyme that becomes activated once the aggregations of platelets has occurred
(Cochrane, 1978). Hageman factor XII is involved in the activation of a series of
downstream conversions in activating pro-enzymes to enzymes that cleaves pro-thrombin
to thrombin, and the final conversion of soluble fibrinogen to insoluble fibrin (Radnoff
and Saito, 1975). In the presence of damaged tissue, the involvement of the extrinsic
pathway includes the release of a lipoprotein, also known as tissue factor, which is found
expressed on activated monocytes and endothelial cells (Kjalke et al., 2000).
The involvement of platelets is also important in other detailed aspects of wound
healing such as re-epithelialization, fibroplasia, and angiogenesis (Kirsner and Eaglstein,
1993). The fibrin clot serves as a temporary scaffold for migrating leukocytes,
keratinocytes, fibroblasts and endothelial cells, in addition to acting as a reservoir for
growth factors. Additionally, the platelets release chemotactic factors such as TGF- and
TGF , which attract leukocytes to migrate into the wound (Wardlaw et al., 1986).
6
1.3.2 Inflammation: The Cellular Response
Acute inflammation is defined by the infiltration of growth and cytokine signals
needed to direct the movement of a variety of inflammatory cells needed for tissue repair.
In addition to blood clotting and the coagulation cascade, chemoattractants such as
monocyte chemoattractant protein-1 (MCP-1), and the presence of bacterial products like
lipopolysaccharides attract more neutrophils (a subset of polymorphonuclear [PMN]
leukocytes) to the site of injury (Balamayooran et al., 2011). The presence of
proinflammatory cytokines such as IL-1 , TNF- , and IFN- , is essential for the
activation of adhesion molecules including: endothelial P- and E-selectins, ICAM-1 and -
2 on endothelial cells. These adhesion molecules allow neutrophils to adhere using
integrins such as CD11a/CD18 (LFA-1) and transmigrate through the blood vessel wall; a
process known as diapedesis (Schubert et al., 1989).
The role of neutrophils is to phagocytose and kill foreign infectious agents through
the release of highly reactive oxygen species (ROS) that damages the DNA of bacteria
through oxidation (Wright et al., 2010). Other antimicrobial substances that aid in this
process include cationic peptides, eicosanoids and proteases like elastases and cathepsin
G (Kaplan, 2011). Neutrophils are also required for the subsequent activation of
myofibroblast differentiation (Peters et al., 2005). According to Peters and coworkers,
CD-18 (a neutrophil integrin) knock-out (KO) mice show delayed wound healing as a
result of impaired myofibroblasts differentiation, leading to poorly closing and
contracting open wounds. The authors suggest that the absence of neutrophils prevents
the release of transforming growth factor β-1 (TGF -1) by macrophages. TGFβ1 is a
proinflammatory cytokine that is necessary for mediation of myofibroblast differentiation
(Desmoulière et al., 1993; Rønnov-Jessen and Petersen, 1993).
Once neutrophils are no longer needed, they are phagocytosed by macrophages
which arrive approximately two days post injury. Macrophages which are derived from
monocytes (another subset of PMNs), are recruited to the site of injury from the blood in
the presence of a variety of chemotactic factors. These factors include proinflammatory
cytokines and chemokines (such as MCP-1) released from platelets, hyperproliferative
keratinocytes at the wound edge, fibroblasts, and leukocytes subsets (Fujiwara and
7
Kobayashi, 2005). The entry of monocytes from the blood vessels into the site of tissue
injury occurs through the interaction of integrin , and potentially 7, endothelial
vascular cell adhesion molecule-1 (VCAM-1). Changes in gene and subsequent
phenotype expression result in the transformation of monocytes into mature tissue
macrophages (van der Rhee et al., 1979). In addition to performing phagocytosis,
macrophages play a major role in activation of other inflammatory cells such as T
lymphocytes by presenting foreign antigens using their cell surface toll-like receptors,
complement receptors and the Fc receptors. Moreover, macrophages secrete a variety of
growth factors, including TGF- , TGF -1, basic fibroblast growth factor (bFGF),
platelet-derived growth factor (PDGF), and vascular endothelial growth factor (VEGF).
These growth factors promote cell proliferation, synthesis of ECM molecules by resident
skin cells, and ultimately the fate of wound healing response (DiPietro, 1995).
The last two inflammatory cells that are involved in the cellular response are the
mast cells and T cells. Although much less emphasized, mast cells are also part of the
PMN subset that function to secrete a variety of proinflammatory mediators and
cytokines that aid in the promotion of inflammation and vascular changes. Mast cells
decrease in number forty-eight hours after injury and increase again once tissue repair
occurs (Theoharides et al., 2012). Once wound closure has occurred and local infections
have been conquered, T lymphocytes are found most abundantly in the wound
(Engelhardt et al., 1998; Fishel et al., 1987). Lymphocyte chemotaxis and accumulation
are associated with the initial appearance of MCP-1which arises four days post injury by
the presence of both chemokine IFN- -inducible protein-10, and monokine induced by
IFN- supplied by macrophages. The deletion of the IFN- gene has been shown to
accelerate the wound healing response, particularly by enhancing the levels of TGF -1 at
the wounding site, and subsequently leading to an increase in collagen deposition (Ishida
et al., 2004).
1.4 Remodelling Phase: Formation of Granulation and Scar Tissue
Around four days post injury a new stroma begins to form (Fig. 1b). The lack of
oxygen resulting from damaged tissue stimulates the release of acidic and basic FGFs
8
from macrophages, as well as VEGFs from the epidermal cells (Eming et al., 2007). The
development of new capillaries (neo-vascularization) provides the oxygen and nutrients
required by infiltrating cells to maintain their metabolism and to sustain the newly
formed granulation tissue. Macrophages and fibroblasts deliver the growth factors needed
for fibroplasia and angiogenesis, and a new provisional ECM for cell migration. PDGF
and TGF -1 are two of the most important growth factors that act in concert with the
ECM to stimulate resident fibroblasts to proliferate and express the appropriate integrins
that facilitate their migration into the injured site (Eming et al., 2007). Additionally,
fibroblast movement through the highly crossed-linked fibrin blood clot requires
proteolytic cleavage which is initiated by a variety of fibroblast-derived enzymes
including plasminogen activator, collagenases, gelatinase A, and stromelysin, in addition
to serum-derived plasmin. Once fibroblasts migrate into the wound, synthesis and
deposition of the ECM occurs which subsequently becomes replaced by a collagenous
ECM. Here the collagen ECM is constantly remodelled by matrix metalloproteinases
(MMPs) secreted by macrophages, epidermal, endothelial and fibroblasts (Guo and
Dipietro, 2010).
Remodelling involves controlled ECM degradation by proteolytic activity of at
least two families of enzymes: MMPs and plasmin. MMPs are a large family consisting
of three groups: (1) collagenases which degrade type I to IV of collagen at the interstitial
level, (2) collagenase/gelatinase which degrade collagen and gelatine, and last, (3)
stromelysins which degrade a broad range of substrates including proteoglycans, laminin,
gelatine, and FN (Birkedal-Hansen et al., 1993). These enzymes are produced by a
variety of cells including endothelial, macrophages and fibroblasts and are known to have
different stimulus effects depending on the cell type. Plasmin is also regulated by the
activation of MMPs and is particularly active in the degradation of laminin located in the
basement membrane, and sometimes in the degradation of gelatine, FN and collagen type
III, IV, and V. TIMPs or tissue inhibitors of MMPs prevent the activation of MMPs,
which is necessary for regulating the survival of myofibroblasts during ECM remodelling
(Kirk et al., 1995). For example, applying skin over granulation tissue was found to
involve rapid remodelling of the granulation tissue which was associated with an increase
9
in the level of MMP activity and a decrease in activity level of TIMPs. In addition, during
the rapid remodelling of the granulation tissue there was also a reduction in the level of
growth factor expression, increased in extracellular ECM turnover, and nitric oxide
generation all of which contributed to fibroblast and vascular cell apoptosis in these
tissues (Darby et al., 2002). On the other hand, in a fibrotic liver, higher levels of TIMPs
were found compared to MMPs, which corresponded to inhibiting fibrotic apoptosis
(Arthur, 2000).
During the second week of healing, wound contraction occurs due to a coordinated
interaction of cells, ECM and cytokines. Predominantly, the presence of TGF -1 and
PDGF, the attachment of fibroblasts to the collagen ECM, and the stiffness of the ECM
as a result of cross-linked collagen stimulates the differentiation of contractile
myofibroblasts. The following section is dedicated to these important cells that are
characterized by de novo expression of -SMA, ECM secretion and contraction
(Gabbiani, 1981; Gabbiani, 1984; Gabbiani, 2003; Hinz, 2007; Hinz et al., 2007). Once
the wound is filled with new granulation tissue, fibroblasts stop producing collagen, and
newly formed blood vessels disintegrate as a result of cellular apoptosis. The underlying
mechanism of this process is not clear, however, the presence of ECM molecules in
particular thrombospondin 1 and 2, and anti-angiogenic factors such as angiostatin,
endostatin and angiopoietin 2 is required (Eming et al., 2007). Eventually, granulation
tissue is replaced by an acellular scar, representing the end point of normal wound
healing and repair (Fig.1c.). This protective barrier that is formed on the skin is thought
to be an evolutionary revenge of mammals for their rapid inflammatory response and
ability to heal without infection (Bayat et al., 2003). Scarring varies both quantitatively
and qualitatively among species and in different organs and body sites. A foetus for
example can heal with very little to no scar formation, as a result of the absence of the
fibrotic-scar reactions that normally occurs in adults. Similarly, the oral cavity
phenotypically shows a lack of scarring, similar to the foetus, which has led to the
speculation that the oral fetal fibroblasts are less contractile and are more motile (Larjava
et al., 2011; Szpaderska et al., 2003).
10
The resolution of inflammation is important in limiting the progression of scarring
towards a chronic disease state. This process occurs through a series of events: 1)
removal of stimulus, 2) dissipation of mediators, 3) cessation of cell infiltration, and 4)
clearance of inflammatory cells, mainly through apoptosis (Serhan and Savill, 2005).
Fibrogenesis will continue to persist as long as inflammation continues leading to further
scarring and subsequently organ failure such as pulmonary and liver fibrosis (Fig 1c).
These fibroproliferative diseases are characterized by the excessive amounts of collagen
deposition, including abnormalities in the cell migration, proliferation, inflammation,
synthesis and secretion of ECM proteins and cytokines (Wynn, 2007).
1.5 Fibroblast-to-Myofibroblast Differentiation in Tissue Repair
Fibroblasts are residential cells that exist as heterogeneous populations within the
soft connective tissues of our body. These cells appear stellate and exhibit elongated
branching processes. Using electron microscopy, one can identify the prominent rough
endoplasmic reticulum and Golgi apparatus indicative of high biosynthetic activity that
occurs in these cells (Gabbiani et al., 1971; Ross, 1968). Approximately 40 years ago,
specialized fibroblasts were first reported to be responsible for the synthesis, deposition,
turnover, and contraction of the ECM, all of which are essential to the process of wound
healing and repair (Gabbiani et al., 1971).
1.5.1 Myofibroblast Features
Myofibroblasts are generated in response to a variety of fibrogenic mediators that
are present in the microenvironment. Fibroblasts developing into myofibroblasts undergo
the following sequence of activities: differentiation, migration, proliferation, and
increased synthesis and deposition of the ECM. The term “myofibroblast” first originated
from the observation that these specialized, differentiated fibroblast also expressed
smooth muscle cell features. Three essential elements are required for myofibroblast
differentiation which all act in concert: TGFβ-1, ED-A FN, and a stiff ECM.
TGFβ-1, which is initially produced by local inflammatory cells in response to
injury, stimulates fibroblasts to increase the production of ED-A FN as well as other
11
ECM proteins, such as collagen type I. In addition the presence of TGFβ-1 as a
chemotactic factor for fibroblasts stimulates their migration towards the site of injury and
the formation of microfilament bundles (in vivo stress fibres), initially composed of β-
cytoplasmic actin. The deposition of ECM proteins and the forces generated by migrating
fibroblasts lead to an increase in ECM stiffness (Hinz, 2009; Hinz, 2010b; Hinz and
Gabbiani, 2010b; Hinz et al., 2001b). It is the rigidity of the ECM that determines the size
of the cell’s anchors, which in turn limits the level of tension generated within stress
fibres (Goffin et al., 2006). Once ECM stiffness reaches a certain threshold, α-SMA
becomes incorporated into pre-existing β-cytoplasmic actin stress fibres (Hinz et al.,
2001a). These specialized actin bundles are found to terminate at the myofibroblast
surface in supermature focal adhesions (8 to 30 μm long), in vivo described as fibronexus,
that help connect the intracellular actin with the fibronectin fibrils found in the ECM
through integrins (Hinz, 2006; Tomasek et al., 2002). The increase in contractile activity
generated by α-SMA stress fibres creates a mechano-transduction system that allows the
force to be distributed to the surrounding ECM. This translates into the contraction and
remodelling of the collagen ECM.
1.5.2 Origins of the Myofibroblast
It is well established that fibroblasts differentiate into contractile myofibroblasts.
One of the most intriguing findings of the last decades was that myofibroblasts not only
derive from resident tissue fibroblasts but from a variety of different precursor cells. It is
now recognized that myofibroblasts, depending on their tissue of origin, can be derived
from: resident cell populations, recruited by circulating precursors, and developed
through the EMT process (McAnulty, 2007). Like smooth muscle cells and pericytes,
myofibroblasts can derive from mesenchymal stem cells (MSCs) (Hinz, 2010a).
12
Figure 2: Origins of the myofibroblast.
1.5.2.1 Resident Fibroblasts
In response to tissue injury, resident fibroblasts from tissue sites adjacent to the
wound proliferate rapidly and migrate into the perivascular region where wounding has
occurred. Proof of fibroblasts recruited in to the surrounding skin wounds was shown by
the strong positive stain for markers such as bromodeoxuridine and MMP-13
(collagenase) (Darby et al., 1997; Desmouliere et al., 2003).
1.5.2.2 Circulating Precursors
Fibrocytes are circulating progenitors for fibroblasts and possibly myofibroblasts
that are involved in both wound healing and fibrosis (Bucala et al., 1994). In contrast to
fibroblasts, fibrocytes originate and differentiate from hematopoietic stem cells. Upon
differentiation from monocytes, fibrocytes lose their leukocyte lineage markers such as
CD14 and CD16 and begin to express markers more typically associated with fibroblasts
There are three essential sources of fibrogenic myofibroblasts: resident cells, epithelial to
mesenchymal transition (EMT), and bone marrow (BM) derived. Below are the cell surface
markers that are expressed by different fibrogenic cells: lymphoid markers (CD45, MHCII,
MHCI), myeloid markers (CD11b, F4/80, Gr1), adhesion molecules (CD54 (ICAM-1) CD80,
and CD86) and fibroblastic markers (Thy-1, collagen α1 (I), and -SMA). Reproduced from:
(McAnulty, 2007).
13
in particular collagen I and CD34 (Fig.2) (Strieter et al., 2009). These cells can give rise
to myofibrocytes which are also known to express -SMA upon TGF -1 stimulation
(Hinz, 2010a).
1.5.2.3 Epithelial-to-Mesenchymal Transition - EMT
The process of EMT involves biochemical and cytokine signalling due to the re-
programming that occurs in the tissue epithelium (Quaggin and Kapus, 2011; Thiery et
al., 2009). MMPs are involved in the disassembly of the basement membrane creating a
loss in both cell polarity and cell-to-cell contacts within the epithelium. During this
process, a significant decrease in the expression of epithelial markers such as E-cadherin,
claudins, zona occludens-1, and cytokeratin-18 occurs, while the increase in fibroblast
specific protein-1 (FSP-1), TGFβ-1, EGF, and FGF-2 facilitate the formation of
fibroblasts by binding to epithelial receptors; this process is rapidly enhanced facilitating
the proliferation of fibroblasts required during inflammation (Neilson et al., 2003). As a
result of FSP-1 interaction with epithelial cell receptors, a downstream activation of the
Ras and c-Src pathways causes a shift in the small GTPase activity and the formation of
pseudopodia in epithelial cells to facilitate directional movement. This process is what
allows fibroblasts to migrate and infiltrate the wound area and deposit new ECM (Lee et
al., 2006). The role of EMT in fibrosis is an actively debated topic (Kisseleva and
Brenner, 2011; Zeisberg and Duffield, 2010).
1.5.3 Mediators and Modulators of the Myofibroblast
Fibroblast activity is regulated by a combination of growth factors, soluble
mediators, and the interplay of ECM components and mechanical stress.
1.5.3.1 The Role of Growth Factors in Fibroblast Fibrogenesis
Growth factors and cytokines such as PDGF, FGF-2, and TGF -1 are mainly
produced by resident and infiltrating inflammatory cells, such as macrophages; these
growth factors are also intricately involved in the activity and recruitment of fibroblasts
(Ornitz and Itoh, 2001). In particular, the importance of TGF -1 in mediating
fibrogenesis has been shown in various organs from the skin, to the kidney, and the lungs.
14
A paper published by Border and coworkers showed that the effects of TGF -1 by using
neutralizing antibodies, and the natural TGF -1-binding glycoprotein, decorin, resulted in
abrogating the promotion of renal fibrosis (Border et al., 1990).
The binding of connective tissue growth factor (CTGF/CCN2) is also known to
activate downstream signalling of TGF -1. Secreted by mesenchymal cells, CTGF is
known to induce and modify adhesive signalling both independently and in response to
growth factors and the ECM. Under normal conditions, CTGF is expressed during
embryogenesis and in wound healing, and found to be over-expressed in fibrotic diseases
in which the combination of both TGF -1 and CTGF is known to promote fibrogenesis
(Leask et al., 2009). Angiotensin II and endothelin-1 are two other vasoactive mediators
that are expressed on both fibroblasts and mesenchymal cells that have pro-fibrotic
effects. On the contrary, endogenous factors such as hepatocyte growth factor (HGF),
bone morphogenic factor-7 and relaxin (hormone) are all antagonists of TGF -1, thus
reducing these factors can accelerate fibrotic development (Leask, 2007).
1.5.3.2 Signal Transduction in Fibroblasts
Fibroblasts are able to sense the mixture of inflammatory, fibrogenic and anti-
fibrogenic factors in the microenvironment through receptor binding and non-receptor
mediated signalling. Receptors function to maintain cellular homeostasis by translocating
signals from the extracellular environment to inside the cell. These receptors, known as
integrins, are transmembrane proteins that bind to specific ECM ligands initiating the
formation of focal adhesions and the activation of focal adhesion kinases (FAK) (Aplin et
al., 1998; Aplin et al., 1999). As a result, a downstream signalling cascade leads to a
diversity of biological processes including cell proliferation, migration, and apoptosis.
Integrins are heterodimer in structure composed of paired and chains (Brakebusch
and Fassler, 2003). Thus far, 14 and 8 subunits have been recognized, with pairings of
either 1 or v chains known for their particular role in binding and adhering to the ECM
proteins (Humphries et al., 2006) . For example, 5 1 integrins are well studied, and
bind to the arginine - glycine - aspartate (RGD) amino acid sequence located in the FN
15
fibrils and other ECM proteins which make up part of the ECM (Danen and Yamada,
2001; Wu et al., 1993).
TGF -1 is an essential fibrogenic factor that signals through the serine/threonine
kinase pathway (Atfi et al., 1995; Laping et al., 2002). Activation of the TGF -1 receptor
results in the rapid recruitment and phosphorylation of the cytosolic Smad protein,
Smad3, which then complexes with Smad4 and translocates into the nucleus where they
regulate the expression of target genes (Fig.3). Although both Smad2 and Smad3 have a
92% homology, they do not share similar DNA-binding activity (Moustakas et al., 2001).
In addition, overexpression of Smad3, and not Smad2 was found to increase TGFβ-1-
induced α-SMA promoter activity and α-SMA protein expression in vitro suggesting, that
TGFβ-1/Smad3 is a major pathway involved in the regulation of myofibroblast
differentiation (Gu et al., 2007; Hu et al., 2003).
16
Figure 3: TGFβ-1-induced-α-SMA transcription in myofibroblasts
The myofibroblast cytoskeleton can function as a mechano-transducer translating
force into biochemical signals involving the tyrosine phosphatase and kinase pathways
(Dallon and Ehrlich, 2010). Mechanical force-induces the p38 rhoA stress fibre-
dependent pathway and a feed-forward amplification loop is used to synergize the force-
induced α-SMA expression with p38 activation (Hu et al., 2006). Cell adhesion signalling
via FAK may represent another central pathway through which biochemical and
biophysical ECM signals as well as soluble growth factor signals are integrated. The
main myofibroblast inducer, TGFβ-1, is known to up-regulate the expression of FN and
TGF -1-induced -SMA expression in myofibroblasts is mediated by Smad3 activation and
subsequent association with Smad4 which forms a complex with a variety of transcription
factors (TF) which all together translocate into the nucleus. IFN- is an inhibitor of -SMA
transcription by up-regulating the expression of Smad7. Krüppel-like factors Sp1/3 enhance -
SMA transcription, and the binding of the TEF-1 to the MCAT-1 element is crucial for -SMA
expression in myofibroblasts (Reproduced from (Hinz, 2007))
17
its corresponding integrin receptors such as 5 1 integrin in fibroblasts (Moir et al.,
2008). This subsequently leads to the activation/phosphorylation of FAK, which is
essential for the inducing myofibroblast differentiation (Lowrie et al., 2004). In addition
to receptor binding, non-receptor mediated signalling such as through nitric oxide has an
influence on fibroblast function (Schaffer et al., 1997).
1.6 The Myofibroblast ECM
The ECM also has a regulatory role on fibroblast activity such as proliferation and
collagen synthesis which are mediated by integrins and their ability to adhere to the ECM
(Darby and Hewitson, 2007). Paine and Ward have shown that when lung fibroblasts
adhere more firmly to the ECM in normal conditions, these cells are more likely to
undergo proliferation and synthesize more collagen. Fibrotic fibroblasts on the other
hand, continue to proliferate in the presence of soft substrate in an adhesion-independent
manner (Paine and Ward, 1999). Thus, the ECM does play a significant and influential
role in the maintenance of tissue homeostasis. I will here concentrate on FN biology and
the role of the ED-A FN splice variant in fibrosis, which is the main topic of my thesis.
1.6.1 Fibronectins (FNs)
FNs are major constituents of the ECM of tissues and plasma. FN is a dimer
composed of two 220-240 kDa homologous (but non-identical) glycoproteins that are
linked via disulfide-bonds (Bae et al., 2004). Each strand expresses multiple binding
domains towards ECM proteins such as collagen and fibrin, as well as for specific
integrins that promote proper cellular attachment (Fig. 4) (Muro et al., 2003). The
adhesive role of FN not only makes this ECM protein an excellent substrate for cellular
adhesion, but also for migration and spreading of diverse cell types during
embryogenesis, cytoskeleton organization, phagocytosis, haemostasis, tumour invasion,
host defense, wound healing, and for maintaining tissue integrity (ffrench-Constant,
1995; Kornblihtt et al., 1996; Serini and Gabbiani, 1999b). The importance of FN was
shown under in vivo conditions where FN-null mice died early during the embryonic
stages of development (George et al., 1993). Clinical studies demonstrated that patients
with mutated FN genes developed glomerulopathy (Castelletti et al., 2008).
18
Figure 4: Cartoon schematic of one arm of FN
FN was one of the first genes reported to undergo alternative splicing, although now it is
estimated that up to 60% of all genes undergo some alternative splicing (Kornblihtt et al.,
1984; Schwarzbauer et al., 1983). From a single gene, three major splice variants are
generated: ED-A, ED-B, and IIICS. Both ED-A and ED-B FN are type III modules that
are each encoded by a single exon which is either excluded or included in the mature
mRNA. The IIICS region, also known as the variable or V region (V0, 64, V89, V95, and
V120), is non-homologous to the other FN domains (Bae et al., 2004), and can undergo
further intricate splicing varying in number depending on the species, i.e. five in humans,
three in rodents and two in frogs (White et al., 2008). As a result of alternative splicing,
up to twenty different sub-variants of FN mRNA transcripts can result depending on
tissue specificity and on the disease state (Muro et al., 2003).
Two main types of FN are generated as a result of alternative splicing: plasma FN
(pFN) and cellular FN (cFN). pFN is a disulphide-bonded dimer that lacks both the ED-A
and ED-B domains but retains the V region variability. This soluble form of FN is found
circulating in the blood and mainly produced by hepatocytes (Xia and Culp, 1995). In
comparison, cFN is a cross-linked multimeric protein that includes the ED-A and/ or ED-
B domains, and is synthesized by a variety of cells including: fibroblasts, mesenchymal,
epithelial and inflammatory cells. CFN are insoluble fibrils due to the ability to form
additional covalent bonds, particularly intermolecular disulphide bonds that cross-link
with other FN dimers. These stabilized fibrils are deposited into the ECM (Williams et
al., 1983).
FN is a dimer consisting of two homologous but non-identical chains held together at the C-
terminus by disulphide bonds. Full details are explained in the body text.
19
1.6.2 ED-A Fibronectin (ED-A FN)
The ED-A domain of FN has been implicated in many functions which include cell
attachment and migration (Manabe et al., 1999; Xia and Culp, 1995), tissue repair (Clark
et al., 1983; Ffrench-Constant and Hynes, 1988), ECM assembly (Guan et al., 1990), FN
dimer formation (Peters et al., 1990), protein secretion (Wang et al., 1991), cytokine-
dependent matrix metalloproteinase expression (Satoi et al., 1999), cell differentiation
(Jarnagin et al., 1994; Serini and Gabbiani, 1999b), tissue injury and inflammation
(Okamura et al., 2001; Satoi et al., 1999), cell cycle progression and mitogenic signal
transduction (Manabe et al., 1999). Here, I discuss the current understandings on the
structure and role of FN splice variant, ED-A FN, particularly in the context of wound
healing and fibroproliferative disorders.
FNs are normally compact in structure held strongly together by both intra- and
intermolecular bonds (Benecky et al., 1990; Erickson and Carrell, 1983). One of the ways
that FN can unfold is by the incorporation of ED-A domain into the mature mRNA. ED-
A domain insertion occurs between two cell adhesion binding domains: the cell central
binding domain (CCBD) and the COOH-terminal heparin-binding domain (Hep2), which
causes an 180° rotation in the regions between the NH2 terminus to the III11 module of
the FN molecule with respect to the COOH terminus of the ED-A domain. As a result of
the change in confirmation, cryptic sites such as the RGD motif serve as an essential site
for integrin recognition and binding, and the synergy sequence, proline-histidine-serine-
arginine-aspartate (PHSRN), which optimizes the binding between the integrin and the
RGD motif, are both revealed. Both these cryptic sites are found hidden within the CCBD
of FN (Benecky et al., 1990; Manabe et al., 1997).
The presence of ED-A FN has been specifically shown to enhance the binding of
integrin α5β1 to the RGD sequence of FN (Manabe et al., 1997). This association leads to
the activation of the FAK pathway which is not only involved in mediating cell
attachment, but also in cellular proliferation by inducing the transition from the G1 to the
S phase of the mitogenic cycle. The activation of ERK MAP kinase in the FAK pathway
plays a major role in enhancing the activation of p38 Src kinase, a downstream molecule
20
in the TGFβ/SMAD signalling pathway, which subsequently leads to an up-regulation of
α-SMA, a characteristic indicative of myofibroblast differentiation (Ding et al., 2008).
1.6.3 ED-A FN Expression
In normal adult tissues, ED-A and ED-B FN are expressed at low levels. However,
during embryogenesis or in pathological situations such as inflammatory diseases, wound
healing, vascular intimal proliferation, cardiac transplantation, and in invasive tumours,
the level of cFN, especially ED-A FN, is up-regulated (Satoi et al., 1999). Although the
role of ED-A FN in wound healing still remains elusive, there is a defined relation
between the anti-inflammatory pro-fibrotic cytokine, TGFβ-1, and the level of ED-A FN
expression. As shown by Hinz and coworkers as well as Serini and coworkers, the
presence of active TGFβ-1, similar to an inflammatory response, significantly enhances
the synthesis of ED-A FN by myofibroblasts, in comparison to normal physiological
conditions where active TGFβ-1 and ED-A FN expression are present at minimal levels
(Hinz et al., 2001a; Serini et al., 1998a).
1.6.4 Generation of ED-A FN Occurs through Alternative Splicing
Production of ED-A FN is generated through alternative splicing which occurs
during embryogenesis and decreases as development continues. However in adults,
similar embryonic FN splice patterns occurs during wound healing, fibrotic disease and
angiogenesis, where ED-A FN levels are enhanced. The incorporation or exclusion of
ED-A domains into the mature mRNA is regulated by serine-and arginine-rich (SR)
proteins (Lavigueur et al., 1993). The pre-mRNA of FN contains a purine-rich region,
known as exonic splicing enhancer (ESE) that is displayed on the loop region of a stem-
loop structure attached to the ED-A exon (Fig. 5); one of the first exons reported to carry
an internal regulatory sequence (Mardon et al., 1987). The ESE region is recognized by
an SR protein, SF2/ASF, and upon binding, the interaction causes the stabilization of the
secondary structure. Therefore the ED-A exon is included in the mature mRNA, resulting
in the formation of ED-A FN. An inhibitor of SF2/ASF is the heterogeneous nuclear
ribonucleoprotein, hnRNP A1, which prevents SF2/ASF from recognizing the ESE. As a
21
result, the ED-A domain is excluded from the mature mRNA, thereby forming pFN
(White et al., 2008).
Although the exact mechanisms involved in triggering ED-A FN alternative
splicing are not known, a novel study by White and coworkers suggests the involvement
of the tumour suppressor “phosphatase and tensin homolog deleted on chromosome 10”
(PTEN) in regulating the inclusion of the ED-A domain into FN (White et al., 2010).
PTEN is a lipid-based phosphatase that binds specifically to phosphatidylinositol-3, 4, 5-
triphosphate, and antagonizes the PI3K/Akt pathway. This protein is also involved in
dephosphorylating FAK and Src-homology 2 (SH2)-containing proteins which causes a
decrease in both cell migration and growth, and increases apoptosis. PTEN is found
downstream of TGFβ signalling, and is expressed inversely to TGFβ levels. By using
PTEN null fibroblasts and PTEN siRNA treated human lung fibroblasts, ED-A FN
expression levels were elevated, suggesting that PTEN levels found in patients affected
with fibroproliferative diseases, may be the initiating factor in regulating the expression
of ED-A FN (White et al., 2006).
1.6.5 Integrin(s) Associated with ED-A FN
Integrins α9β1 and α4β1 specifically recognize the EDGIHEL motif found within
the ED-A domain. Integrin α9β1 is normally expressed in the respiratory epithelium, the
Figure 5: Mechanisms of ED-A through Alternative Splicing.
Figure 5. Mechanisms of ED-A through Alternative Splicing.
ED-A inclusion into the mature mRNA requires recognition of the exon splicing enhancer
(ESE) by splicing factor SF2/ASF. SF2/ASF must compete with hnRNP A1 which favors the
exclusion of the ED-A exon from the mature mRNA. Similarly, ED-A exon skipping also
occurs in the presence of weak splice sites (Reproduced from (White et al., 2008)).
22
basal layer of squamous epithelia, smooth and skeletal muscle (Palmer et al., 1993; White
et al., 2006) and in cell specific types, including neutrophils, hepatocytes, and
keratinocytes which are known to up-regulate α9β1 during embryogenesis and tissue
repair (Shinde et al., 2008). Cultured fibroblasts and myofibroblasts only express integrin
α4β1 which is known to promote cellular adhesion to the ED-A domain of FN (Gailit et
al., 1993). However, the signal transduction pathway that is activated upon the interaction
between integrin α4β1 and ED-A FN is still currently not known. It should be noted that
integrin α4β1 expression has only been demonstrated in cultured fibroblasts, and is
normally found absent in skin fibroblasts in vivo (Carter et al., 1990; Konter et al., 1989).
This may be reflective of very low expression levels found under normal physiological
conditions which only upon fibroblast activation become up-regulated in situations such
as wound repair and in inflammatory diseases (Gailit et al., 1993).
A recent study by Kohan et al., 2010 provided a link between α4β7, an integrin
shown to bind to the ED-A domain of FN, and myofibroblast differentiation and activity.
By blocking the interaction between α4β7 receptor and ED-A FN, mice lung
myofibroblasts displayed a significant reduction several ED-A FN regulated activities
which included: the ability to adhere to ED-A FN, up-regulate the expression α-SMA,
collagen deposition and FAK activation. In addition, the interaction between α4β7 and
ED-A FN was shown to activate MAPK-Erk1/2 pathway; a pathway involved in
fibroblast differentiation. Hence the importance of this study provides a deeper
understanding, particularly in defining the mechanism of how EDA-FN is involved in
fibroblast differentiation and function (Kohan et al., 2010).
1.6.6 ED-A FN as a Pro-Fibrotic Factor
Numerous homeostatic functions ranging from cellular adhesion and ECM
assembly, to wound healing and mitosis have been ascribed to ED-A FN. Mice that lack
the ED-A module of FN have been well documented for their shorter lifespan (two
months shorter compared to the wild-type), abnormal wound healing, and edemantous
granulation tissue as a result of deregulation in the re-epithelialization process and
ongoing proliferation of infiltrating inflammatory cells that occurs at the wound site
23
(Muro et al., 2003). Yet when deregulation at the translational level occurs, elevated
amounts of ED-A FN serves as a pathogenic factor found in many common
fibroproliferative diseases including psoriasis, rheumatoid arthritis, diabetes and cancer
(Muro et al., 2008; White et al., 2008). Many studies support the presence of ED-A FN as
an essential factor in fibrosis, along with TGFβ-1 and mechanical stress, in myofibroblast
differentiation.
Although many studies report the presence of elevated ED-A FN levels in plasma
and tissues of affected patients, the exact role of ED-A as a pathological factor in diseases
particularly psoriasis, rheumatoid arthritis, diabetes, and cancer, still remains elusive
(George et al., 1993). Recent evidence supporting the role of ED-A FN during wound
healing was shown in vivo, where ED-A-/-
were all associated with abnormal wound
healing, ulceration and inflammation at the wound site (Muro et al., 2003). Deregulation
of ED-A FN affects myofibroblast differentiation which can eventually lead to fibrotic
development and mortality in those untreated (White, 2006). This has been clearly
demonstrated in patients suffering from life threatening fibrosis that affects the lungs,
termed idiopathic pulmonary fibrosis (IPF) (Thannickal et al., 2004; White et al., 2003).
Notably, EDA-/-
lung fibroblasts produce equivalent amounts of TGFβ-1 compared
to the wild-type, and exhibited no defects in the activation of the SMAD signalling
cascade associated with the binding of exogenous TGFβ-1(White, 2006). These EDA-/-
lung fibroblasts however were less capable of activating latent TGFβ-1 from the ECM,
which in turn affected collagen synthesis and α-SMA up-regulation. This observation
could explain why EDA-/-
mice were protected from bleomycin-induced fibrosis (Muro et
al., 2003). However, the components involved in regulating ED-A FN expression and the
exact role that ED-A FN plays in up-regulating these myofibroblastic features i.e. α-SMA
and collagen type I production, are still under investigation. Revealing and understanding
these undefined links serves to properly steer future regenerative medicine towards
development of plausible therapeutic treatments that target ED-A FN.
24
1.6.6.1 Lung Fibrosis
Patients suffering from IPF eventually die of a deregulation in the deposition of
ECM proteins in the lungs (White et al., 2008). In locations of active fibrosis, the
increase in myofibroblast differentiation correlates with the up-regulation of ED-A FN
levels, which was shown to precede the synthesis and deposition of collagen (White et
al., 2008) (Kuhn et al., 1989). The molecular effects of ED-A FN on fibrotic development
was studied by Muro and coworkers, who analyzed the effects on TGFβ-1 activity and
myofibroblast differentiation in ED-A-/-
mice treated with high doses of bleomycin, a
chemical that induces fibrosis development. In these mice, fibrosis was prevented
signifying the intimate association between ED-A FN and IPF development. Surprisingly,
the TGFβ-1/ SMAD signalling pathway was not affected, although the signal
transduction was not as robust as the wild type; there was also no effect on the amount of
latent TGFβ-1 produced by these ED-A-/-
lung myofibroblasts. However the ability to
activate latent TGFβ-1 was significantly affected, which was followed by a reduction in
collagen deposition and α-SMA expression.
To show that ED-A FN is an important factor involved in TGFβ-1-induced
myofibroblast differentiation and collagen expression, rescue experiments were done by
plating ED-A-/-
lung fibroblasts on ED-A FN coated substrates. With the addition of
exogenous TGFβ-1, ED-A-/-
lung fibroblasts expressed higher levels of α-SMA compared
to those plated on tissue culture plastic or ED-A FN alone. Surprisingly, the presence of
both TGFβ-1and ED-A FN induced a lower production of collagen compared to ED-A
FN alone, suggesting that ED-A FN has a regulatory role in post-translational expression
of collagen. The presence of ED-A FN was also able to recover TGFβ-1 activity, which is
an important cytokine involved in regulating the transition from inflammation to fibrosis.
These results draws an important connection between ED-A FN and the role it plays in
the development of pulmonary fibrosis, particularly by the activation of latent TGFβ-1,
myofibroblast differentiation and collagen type I production (Muro et al., 2008).
25
1.6.6.2 Dupuytren’s Disease
Dupuytren’s disease is a chronic inflammatory disease characterized by a lesion of
palmar fascia that immobilizes the flexion of digits. The difficulty in the contraction of
the palmar fascia is a result of active remodelling and turnover, where the ECM is
continuously shortened (Hinz and Gabbiani, 2011). The composition and the shortening
of the ECM during the active and contractive phases of Dupuytren’s disease is similar to
the ECM composition found during the contraction phase of wound healing and
embryogenesis, specifically with a high level of ED-A FN present (Halliday et al., 1994).
The nodules that are present in the hand precede fibrosis development and
contraction of the fingers. Dissection of these nodules reveals the presence of α4β1-
expressing inflammatory cells such as macrophages and T-lymphocytes that bind
specifically to the VCAM-1 ligand expressed on the endothelium of blood vessels (Meek
et al., 1999). The interaction between integrin α4β1 and VCAM-1 enables these immune
cells to transmigrate through the endothelium of the blood vessels and into the blood
stream towards the site of inflammation. As mentioned above, integrin α4β1 also has an
affinity towards the ED-A FN domain which influences the type of growth factors and
cytokines that are subsequently secreted by these inflammatory cells upon binding. These
factors include: bFGF, interleukin-1 (IL-1) α and β, tumour necrosis factor α, IL-8, and
TGFβ-1, which are all known to an influence myofibroblast migration, proliferation, and
contraction (Meek et al., 1999). As a result of chronic inflammation that characterizes
this disease, fibrosis development is inevitable.
1.6.6.3 Atherosclerosis
Under normal conditions, the FN that surrounds the arterial wall expresses very low
levels of ED-A and ED-B splice variants. However, during atherosclerosis the thickening
of the aorta is accompanied by elevated levels of ED-A FN and smooth muscle cell
proliferation (Glukhova et al., 1989; Magnusson and Mosher, 1998). The role of ED-A
FN expression in atherosclerosis is not known, however an increasing amount of
evidence suggests that ED-A domain acts as a ligand for the toll-like receptor 4 (TLR4)
present on inflammatory cells. As a result, the activation of TLR4 leads to the
26
amplification of an immune response and subsequent progression towards atherosclerosis
development (Xu et al., 2001). Interestingly, the same TLR4-ED-A FN interaction was
shown to be responsible for the progression of rheumatoid arthritis (van de Loo and van
den Berg, 2009). Hence, therapeutic treatments that target the expression of ED-A FN
provide a plausible mechanism to help attenuate the pathogenesis of fibroproliferative
disorders (Fig. 4) (Serini et al., 1998b).
1.6.7 The Role of ED-A FN in Myofibroblast Differentiation
Many studies have defined the importance of myofibroblasts in normal wound
regeneration, and in the pathological development of fibrotic diseases such as pulmonary
fibrosis and the stroma reactions to epithelial tumours. The regulatory factors that govern
myofibroblast differentiation are intimately associated, these factors include: ED-A FN,
TGFβ-1 and mechanical stress (Serini and Gabbiani, 1999b).
During a post-wound response, inflammatory cells such as macrophages, release
large amounts of latent TGFβ-1 that not only amplifies the immune response, but also
acts as a chemoattractant for fibroblast migration into the wound site and ED-A FN
secretion (Fig. 1). In addition to the presence of exogenous TGFβ-1 and ED-A FN, the
increase in mechanical tension as a result of an increase in ECM stiffness, altogether
induce fibroblasts differentiation into α-SMA expressing myofibroblasts. The exact role
of ED-A FN governing myofibroblasts differentiation is currently not defined however as
shown by a number of works, ED-A FN is an essential factor required for TGFβ-1-
induced α-SMA expression. This was clearly depicted by using IST-9, an ED-A domain
specific blocking antibody, which resulted in the down-regulation of α-SMA expression
in rat subcutaneous myofibroblasts, even in the presence of TGFβ-1 (Hinz et al., 2001a;
Serini et al., 1998b). It should also be emphasized that both the presence of ED-A FN and
TGFβ-1 is required to promote α-SMA expression and collagen type I production, in
which ED-A may serve as an intermediate factor in the transduction and/ or coordination
of the signals initiated by TGFβ-1 (Serini et al., 1998b).
To date, the interplay between ED-A FN expression and myofibroblast
differentiation still remains elusive. As mentioned above, elevated levels of ED-A FN
27
corresponded to low levels of PTEN in IPF affected patients (Muro et al., 2008). This is
associated with a previous study also done by White and coworkers in IPF patients where
the presence of PTEN negatively regulated the expression of α-SMA, myofibroblast
proliferation and subsequent collagen type I production (White et al., 2006). Although the
connection between PTEN and the regulation of ED-A FN expression still remains
undefined, it remains clear that ED-A FN is a crucial factor in myofibroblast
differentiation.
The results presented in my thesis indicate an intimate associate between LTBP-1, a
protein involved in storing latent TGFβ-1 in the ECM, and ED-A FN. Both these protein
levels are currently being observed for their differential expression and colocalization as
a function of substrate stiffness.
1.7 The Link Between TGFβ-1 and the ECM
1.7.1 The Activation of Latent TGFβ-1
The association of TGFβ-1 with its latency pro-peptide LAP forms the small
latency complex (SLC) that keeps TGF -1 latent (Dallas et al., 2000). LTBP-1 is
responsible for storing latent TGF -1 in the ECM. LTBP-1 forms a covalent bond with
SLC within the cell forming the large latent complex (LLC) which is then deposited into
the ECM. It is only through the dissociation from LAP that mature TGF can bind to its
signalling receptors, which could occur through proteolytic cleavage of LTBP-1 such as
with plasmin at the N-terminus, or release of the whole LLC complex at the weak-
binding region at the C-terminus of LTBP-1. Several cellular mechanisms have been well
documented in the release of latent TGFβ-1 by promoting its dissociation from LAP.
These activation processes include cleavage of LLC though enzymatic associated
mechanisms, specifically proteases such as plasmin and thrombospondin (Jenkins, 2008;
Wipff et al., 2007).
According to Wipff and coworkers, myofibroblasts can activate TGFβ-1 from
LTBP-1 in the ECM in a contraction-mediated manner. There are three mandatory factors
that are required to liberate latent TGFβ-1 from this ECM: 1) high contractile activity
28
generated by α-SMA positive stress fibres; 2) stress transmission to LAP-TGFβ-1 via
integrins; and 3) integration of the LLC into a mechanical resistant ECM (Annes et al.,
2004; Annes et al., 2003; Wipff et al., 2007). The high contractile activity generated by
α-SMA in stress fibres is transmitted at sites of integrins binding to RGD sites in the LAP
protein as part of the LLC, which also includes TGFβ-1 and LTBP-1. When the LLC is
anchored in a stiff ECM, the transmission of cell-mediated stress through integrins
induces conformational changes in the LAP, which subsequently results in the liberation
of active TGFβ-1 and binding to its TGFβ receptor located on the cell surface (Fig. 6). In
the presence of a compliant ECM (Fig. 6), the LLC is dragged in the direction of the
pulling cell, however due to the lack of a mechanically resistant ECM, no conformation
change occurs and TGFβ-1 remains in its latent form bound to LAP (Buscemi et al.,
2011a; Shi et al., 2011). Inhibition in the activation of latent TGFβ-1 could also occur by
interfering with the interaction of integrins from binding to the RGD sequence in LAP.
Figure 6: Model of myofibroblast contraction-mediated TGFβ-1 activation.
Figure 6: Model of myofibroblast contraction-mediated TGFβ-1 activation.
The high contractile activity generated by α-SMA in stress fibres is transmitted at sites of
integrins binding to RGD sites in the LAP protein as part of the LLC, which also includes
TGFβ-1 and LTBP-1. Left panel: When the LLC is anchored in a comparably stiff ECM, cell-
mediated stress can induce allosteric changes in LTBP-1 and/or LAP conformation, leading to
liberation of TGF-β1; such activated TGFβ-1 possibly feeds back by binding to its receptor,
which is located close by in the activating cell. Right panel: In the context of compliant ECM,
the LLC is dragged toward the pulling cell but because of the lack of mechanical resistance, no
conformation change occurs and TGFβ-1 remains latent. Likewise, inhibition of high cell
contraction and interaction of integrins with the LLC block mechanical activation of latent
TGFβ-1 (Reproduced from (Wipff and Hinz, 2008)).
29
To demonstrate the importance of a stiff ECM in association with LTBP-1 in the
activation myofibroblast differentiation, transformation of murine dermal fibroblasts to
overexpress the SLC alone was not enough to induce myofibroblast differentiation
(Campaner et al., 2006). Thus the hypothetical model involving tension-mediated
activation of TGFβ-1 through integrins is a possible explanation of how myofibroblast
differentiation could persist in the presence of a mechanically stiff environment during
wound healing (Goffin et al., 2006) fibrotic lesions (Hinz, 2007), and in the stiff stroma
that surrounds epithelial tumours (Amatangelo et al., 2005; Paszek et al., 2005).
1.7.2 Latent Transforming Growth Factor Binding Proteins (LTBPs)
Interestingly, 10-90% of LTBP-1 can also be secreted in free form lacking the
presence of TGF , and this is dependent on the cell type and the stage at which
differentiation is analyzed (Dallas et al., 2000). The role of the free LTBP-1 is currently
unknown, however this suggests that their role may be independent of storing TGF and
is involved in other ECM related properties. LTBPs are high molecular weight
glycoproteins that are found in the ECM. LTBPs belong to the fibrillin/LTBP-
superfamily composed of EGF-like repeats and 8-Cystein repeats (Unsold et al., 2001).
Currently, four isoforms of LTBP have been described, all of which display 1-2 RGD
sequences for cell adhesion except for LTBP-3 (Gibson et al., 1995; Giltay et al., 1997;
Kanzaki et al., 1990; Moren et al., 1994; Saharinen et al., 1998; Yin et al., 1995).
Two forms of LTBP-1 exist: long and short form denoted by LTBP-1L, and LTBP-
1S, respectively. LTBP-1S consists of 18 EGF-like repeats, 15 of which are Ca+2
-binding,
and 4 of the 8-Cystein repeats, with the first being a hybrid domain (Ramirez and Rifkin,
2009; Todorovic et al., 2005). Similarly to LTBP-1S, LTBP-1L in addition has a 4-
Cystein repeat and a Ca+2
-binding EGF like repeat located at the N-terminal (Koski et al.,
1999). Although both forms can bind to the ECM, in comparison, LTBP-1L is known to
bind at a higher efficiency. The function of the 8-Cysteine residues has never been
defined, however the third of the 8 Cystein domains in which only LTBP-1, 3, and 4 is
known to bind covalently to LAP (Saharinen et al., 1998). Although the physiological
role of the two LTBP isoforms has yet to be defined, it is hypothesized that their
30
independent promoters and their expression in different tissues creates different
localization patterns of the SLC in tissues.
Currently, there are no known diseases associated with mutations in LTBP-1,
however the importance of LTBP is clearly seen when embryonic lethality was seen in
LTBP-2 null mice (Shipley et al., 2000). Moreover, the symptoms of Marfan’s patients
appear to rely on the inability of LTBP-1 to bind to mutated fibrillins (Charbonneau et
al., 2010; Nistala et al., 2010; Ramirez and Rifkin, 2009; Ramirez and Sakai, 2010).
1.7.3 Association of LTBP-1 with the ECM
So far, LTBP-1 has been found to colocalize and bind with FN, elastin, and
fibrillin-1. Initially, it is believed that LTBP-1 primarly associates with FN, then becomes
incorporated into fibrillin positive microfibrils as in vitro cultures are prolonged (Unsold
et al., 2001). In addition, LTBP-1 has been shown through several studies to bind to FN
through transglutaminase and heparin linkages that form near the N- terminals of LTBP-
1. Hence it is believed that the N terminal is involved in anchoring the LLC to the ECM,
and the C terminal is where the binding is stabilized.
Through immunlocalization studies, FN was shown to assemble into the ECM prior
to LTBP-1 deposition, in which these two proteins have been shown to colocalize (Koli
et al., 2005; Koli et al., 2008; Unsold et al., 2001; Wipff et al., 2007). As cultures are
prolonged, FN and LTBP-1 are found to be in separate fibrillar networks, thus suggesting
that the formation of the FN ECM acts only as temporary scaffold for LTBP-1. Hence,
this was shown in two ways: 1) using a N-terminal FN blocking peptide, the LLC was
inhibited from being incorporated into the ECM, and 2) FN KO mouse embryonic
fibroblasts failed to deposit LTBP-1 in the ECM which was found in high amounts in the
media (Dallas et al., 2005). Providing exogenous FN was able to rescue the fibrillar
formation of FN and the integration of LTBP-1 into the ECM, shown by the
colocalization of the two proteins (Wipff et al., 2007). This suggests that the presence of
FN is required for the initial assembly of LTBP-1 in the ECM, and the continued
presence of FN is necessary for LTBP-1 assembly to continue.
31
The interaction between the assembly of FN and fibrillin-1 are interdependent. As
higher order FN ECM assembly continues, the non covalent interactions occur with other
ECM proteins such as fibrillin. It should be clarified that the assembly of fibrillin-1 into
microfibrils is dependent on the initial presence of FN deposition (Massam-Wu et al.,
2010). It still remains unclear whether the interaction between LTBP-1 and fibrillin-1 is
necessary as one studied showed that fibrillin-1 was not required for LTBP-1
incorporation in association with FN (Massam-Wu et al., 2010). However the presence of
heparin sulphate proteoglycans and the dependence on microfibrils assembly and FN,
may regulate the deposition of LLC and bioavailability of TGF (Kinsey et al., 2008).
1.8 The role of ECM Stiffness in Fibrogenesis
Tissues such as the skin are exposed to mechanical stress and tension on a daily
basis. Nevertheless, fibrosis does not develop on a daily basis. Cells are exposed to
pathological levels of stress when mechano-protective ECM is impaired by injury.
Fibroblasts within injured tissues respond by producing changes in the ECM to help in
the adaptation and maintenance of tissue homeostasis. Mechanical tension and stress are
one of the three essential factors that drive the expression of -SMA, to be incorporated
in the stress fibres of myofibroblasts (Hinz et al., 2001b). This was observed in a simple
experiment where a very low percentage of -SMA positive myofibroblasts was found in
a free-floating collagen ECM (Arora and Mcculloch, 1994). In addition, there was very
little contraction of the surrounding compliant ECM. However, when fibroblasts were
embedded in stressed lattices, the number of -SMA positive myofibroblasts increased
which coincided with a decrease in diameter of the surrounding lattice (Tomasek et al.,
2002). It has been proposed that in vivo the tractional forces generated by fibroblasts
during migration is enough to initiate wound closure, however it is the resistance of the
surrounding ECM that promotes the differentiation of myofibroblast allowing true
contraction and closure of the wound to fully occur (Hinz et al., 2004; Wipff et al., 2007).
Notably, the ECM of fibrotic tissue can be 10-50-times stiffer than the respective normal
connective tissue (Hinz, 2010c).
32
Figure 7: The Young’s Modulus of Tissues.
Tissue stiffness is often expressed as elastic or Young’s modulus with the
dimension Pascal (Pa). The Young’s modulus of elasticity was named after the British
scientist Thomas Young in the 18th
century. The Pa unit of the Young’s modulus is a
pressure unit which expresses the ratio of stress (Pa) over strain (dimensionless) as seen
in the formula below:
The tensile elasticity is defined as the tendency of an object to deform along an axis
when opposing forces are applied along that axis. Stress is the restoring force caused due
to the deformation divided by the area to which the force is applied, and strain is the ratio
The schematic displays the different elastic measurements of tissues and organs in our body.
Reproduced from (Paszek et al., 2005).
Where:
E is the Young's modulus (modulus of elasticity)
F is the force exerted on an object under tension;
A0 is the original cross-sectional area through which the force is applied;
ΔL is the amount by which the length of the object changes;
L0 is the original length of the object.
33
of the change caused by the stress to the original state of the object (Butcher et al., 2009;
Janmey et al., 2007; Janmey et al., 2009).
34
Chapter 2: Materials and Methods
2.1 Preparation of Deformable Silicone Substrates
To produce culture substrates of different stiffness, we used a silicone elastomer.
Polydimethyl silicone (PDMS) base (Dow Corning, Midland, MI) was mixed with curing
agent at pre-determined ratios generating different stiffness of 5 kPa, 100 kPa, and 3,000
kPa. Polymer was deposited on 35 mm and 60 mm TPP and BD Falcon petri dishes, and
spun in a spin caster (Spin 150 Rev. 3.2, Semiconduction Products Systems Europe) to
even distribute the polymer across the plate at 14,000 rpm. Plates were allowed to
polymerize at 65 C for 4 days (Goffin et al., 2006; Wipff et al., 2007). Soft substrates
were coated with collagen (10 g/ml) overnight at 37 C in 1x PBS pH 7.4.
2.2 Culture and Analysis of Fibroblasts on Soft Substrates
Human dermal tissue was received as a gift from Benjamin Alman at Sick Kids
Hospital (Toronto, Canada). Tissue was first treated with 10x antibiotics, then stretched
on to tissue culture plastic where the tissue was incubated with 10% FBS (DMEM, 1%
penicillin/streptomycin) to allow for cells to migrate out of the explant. Human dermal
fibroblasts (HDFs) were then trypsinized (0.25%) and expanded seeding them in T75
flasks. HDFs were seeded at 5,000 cells/cm2 and cultured in 10% FBS (DMEM, 1%
penicillin/streptomycin) for 5 days. HDFs were washed several times with 1x PBS (pH
7.4), and fixed with 3% paraformaldehyde for 10 minutes. Cells were permeabilized with
0.2% Triton X-100 in PBS/Ca+2
for 5 minutes, then stained for ED-A FN (anti-IST-9,
mouse IgG1, Santa Cruz Biotechnology, Inc.), LTBP-1 (mouse IgG1, R&D Systems ),
and -SMA (mouse IgG2a; a gift from G. Gabbiani, University of Geneva). Secondary
antibodies, TRITC-and Alexa647-conjugated goat anti-mouse subclasses IgG1 and IgG2a
(SouthernBiotech Associates Inc., Birmingham, AL), and TRITC-, FITC-conjugated goat
anti-rabbit (Sigma-Aldrich) were used.
35
For Western blotting, 10% SDS-PAGE was used to analyze ED-A FN, -SMA,
vimentin (Sigma Aldrich), and GAPDH (Millipore). 8% SDS-PAGE was used to analyze
LTBP-1 under non-reduced conditions. SDS-Page gels were then transferred to PVDF
membranes using semi-dry transfer technique at 18mAmps/gel, 20V, for 16hrs overnight.
PVDF membranes were probed for the same primary antibodies as immunofluorescence
in addition to vimentin (Sigma Aldrich) and GAPDH (Millipore), HRP-conjugated
secondary antibodies goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, Inc,)
and goat anti-rabbit (Cell Signalling Technology) were used. Signals were detected by
ECL chemiluminescence (Amersham, Rahn AG, Zurich, Switzerland).
2.3 Recombinant FN Peptides and FN Constructs
Polyplus transfection (JetPRIMETM
, France) was used to transfect human
embryonic kidney (HEK) cells with pcDNATM
3.1/V5-HIS TOPO TA expression
vector inserted with genes encoding the full-length soluble FN constructs: ED-A FN, ED-
B FN, ED-A/B, and pFN (a gift from Rebecca Wells). HEK cells were grown in 10%
FBS media (DMEM, 1% pencillin/streptomycin, and 1% fungizone), and kept under
selection with 0.2 mg/ml of G418 (Sigma Aldrich). FN constructs produced into the
media from transformed HEK cells was purified using gelatin-sepharose CL-4B (Sigma-
Aldrich) affinity columns. Several washes were done using 1x TBS, and FN was eluted
with 8 M Urea. Quantification was done at absorbance 280 nm and samples were
dialyzed for 2 days in 1x PBS at 4 C.
Transformed E.coli BL21 that produced HIS-tagged recombinant FN peptides: 11th
,
11th
/12th
, ED-A, 11th
/12th
ED-A (a gift from Dr. Eric White) were cultured overnight in
100 g/ml of ampicillin/LB media at 37 C at 200 rpm. New LB/ampicillin media was
added to overnight cultures and re-incubated at 37 C until an OD of 0.6 was reached.
Final concentration of 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to
the culture to stimulate protein expression, and continued to culture for 4 hours. Pellets
were spun down at 15 minutes for 3,320g and stored at -20 C. For recombinant FN
peptides, pellets were lysed in NPI buffer (50 mM NaH2PO4, 300 mM NaCl, at pH 8),
lysozyme (Sigma L1667), protease inhibitor cocktail for Histidine-tagged proteins
36
(Sigma H77898), and benzonase nuclease (Sigma E10104). Lysate was cleared by
centrifuging at 10,000x g for 20 minutes, and then purified using HIS-select Hi Flow
Cartridges (Sigma Aldrich). Recombinant FN peptides were washed with NPI-10 and -
20, then eluted with NPI-250, and dialyzed overnight in 1xPBS at 4 C. Coomassie
staining was done to check the presence and purification of the peptides.
2.4 Solid Phase Binding Assay
Recombinant ED-A peptides and full FN constructs were reconstituted in 15 mM
sodium carbonate and 35 mM sodium bicarbonate buffer at pH 9.2 and incubated
overnight at 4 C in 96 well microtiter plates (BD Biosciences) at increasing
concentrations of 50 g/ml, 100 g/ml, and 200 g/ml. The wells were then blocked with
5% skim milk in 1x TBS (pH 7.4) for an hour at room temperature. Purified LTBP-1 was
diluted in 2% skim milk/TBS in 2 mM CaCl2 and used at a concentration of 50 g/ml and
incubated for 3 hours. Primary antibody, Ab39, was diluted in 2% TBS milk and was
used to detect LTBP-1 for 2 hours. 3 x 5 minutes washes was done with 0.05% Tween
20/TBS. Secondary antibody was diluted in 0.05M carbonate/bicarbonate buffer at pH
9.6 and incubated for 1 hour. Repeats of the same washes were done. 200 l of p-
nitrophenyl phosphate (Sigma Aldrich) was added to each well and incubate for half an
hour at room temperature in the dark. Absorbance was read at wavelength of 405 nm.
2.5 ED-A Blocking with IST-9 Antibody and Recombinant Peptides
To inhibit the ED-A domain of FN with antibodies, HDFs were seeded at 50,000
cells/cm2 on 1 x 1cm glass coverslips. Cells were then incubated with 300 g/ml of IST-9
(Santa-Cruz) for 4 days. Controls were incubated with 1xPBS. Immunofluorescence and
Western blotting were used to analyze results.
For competitive blocking of ED-A FN with recombinant peptides, HDFs were first
seeded at 50,000 cells/cm2 on 1 x 1cm glass coverslips, and 100,000 cells/cm
2 on 60 mm
dishes, then incubated with 100 g/ml of recombinant FN peptides after 24 hrs of seeding
37
for the next 4 days. Results were analyzed by both immunofluorescence and Western
blotting using the same antibodies as previously described.
2.6 Co-Immunoprecipitation of LTBP-1 and ED-A FN
HDFs were seeded on 60 mm petri dishes for 4 days at 37 C, 5% CO2. Anti-LTBP-
1 and IST-9 antibodies were incubated with protein-G Sepharose beads in a 1:20 ratio
overnight at 4 C. Supernatant was collected and incubated with protein G sepharose
beads at 4 C on a rotator (pre-clearing). Cells were washed 3 x 5 minutes with 1x PBS
(pH 7.4) and incubated with 1% gentle lysis 3-[(3-Cholamidopropyl)-dimethylammonio]-
1-propane sulfonate (CHAPS) buffer (0.1 M Tris, pH 8; 0.5 M EDTA pH 8; CHAPS;
protease inhibitor cocktail) for 30 minutes on ice at room temperature. Cells were then
scraped and total cell lysate was collected and pre-cleared for an hour. Both media and
total cell lysate were centrifuged at 14,000 x g at 4 C for 10 minutes. The bead pellet was
discarded and the residual “purified” supernatant and total cell lysate was kept. Both the
supernatant and total cell lysate was incubated with anti-LTBP-1 and IST-9 coated
protein-G sepharose beads separately O/N at 4 C with gentle agitation, then centrifuged
at 800 x g for 3 minutes at 4 C. Western blot analysis on the supernatant and total cell
lysate was done staining for ED-A FN and LTBP-1.
2.7 LTBP-1 Expression in Wild-type ED-A and ED-A-/-
Mice
Subcutaneous fibroblasts were seeded on TCP for 7 days in 10% FBS (FN
depleted) medium. Medium was collected and immunoprecipitation using anti-LTBP-1
coated protein G sepharose beads was used to compare the amount of LTBP-1 (rbAB39,
a kind gift of Dan Rifkin, New York University) released in the medium in the two
different phenotypes. Immunofluorescence and Western blot were used to observe the
organization and quantify the amount of LTBP-1 deposited into the ECM, respectively.
Wild-type ED-A and EDA-/-
mice were a gift from Kristen Bielefeld and Ben
Alman at Sick Kids Hospital (Toronto, Canada). The dorsal skin was submersed in 10x
antibiotics and the subcutaneous tissue was removed and stretched out on 60 mm tissue
38
culture plastic dishes (BD Falcon) enabling fibroblast migration. When the TCP dishes
reached 90% confluence, fibroblasts were trypsinized at a concentration of 0.25% (in
DMEM), blocked with 10% FBS, centrifuged at 1.5 rpm for 5 minutes, then resuspended
in 10% FBS (1:100 pen/strep) in a T75 flask for cell expansion at 37 C, 5% CO2.
39
Chapter 3: Results
One hypothesis of my thesis is that ED-A FN contributes to the storage of latent
TGFβ-1 in the ECM by interacting with LTBP-1, the TGFβ-1 storage protein.
Accordingly, the first objective of my thesis was to demonstrate that LTBP-1 displays a
preferential and possibly specific binding to ED-A FN. A direct interaction between these
two factors that are essential for myofibroblast differentiation would establish a link
between TGFβ-1 activation and ED-A FN expression in the ECM.
3.1 LTBP-1 binds to ED-A FN in Vitro Mainly in the ECM of HDFs
To first test whether ED-A FN binds to LTBP-1, I performed co-
immunoprecipitation experiments under native conditions. In Western controls performed
with cultured HDF, ED-A FN was found mostly in the total cell lysate (ECM and cells),
while very little was found in the conditioned medium (Fig. 8, left). Analyzing the
Western blot staining for LTBP-1 (Fig. 8, right), controls show that most LTBP-1 was
found present in the ECM, while very little was found in the conditioned medium. Non-
coated beads confirmed that there was no non-specific binding.
Figure 8: Co-immunoprecipitation of LTBP-1 and ED-A FN
The potential binding between LTBP-1 and ED-A FN was analyzed by co-immunoprecipitation
from total cell lysates (including ECM) and media of HDFs. Blots were probed against LTBP-1
(Ab39) and ED-A FN (IST-9).
40
Immunoprecipitation performed with IST-9 coated protein G sepharose beads
detected most ED-A FN in the ECM and cells, while little was found in the conditioned
medium. Interestingly, beads coated with anti-LTBP-1 pulled down ED-A FN showing
that LTBP-1 binds to ED-A FN in the ECM. IST-9 coated protein G sepharose beads
pulled down LTBP-1 confirming that most of the LTBP-1 associated with ED-A FN in
the total cell lysate/ECM. The interaction between these two proteins was also shown to
be present in the conditioned medium at very low levels. This is not surprising since
LTBP-1 and ED-A FN are soluble proteins that are released into the media by the cells.
In conclusion, in the ECM of cultured HDF, LTBP-1 and ED-A FN bind to each other.
3.2 ECM Stiffness affects Co-Expression of ED-A FN and LTBP-1
My second hypothesis was that the levels of ED-A FN and LTBP-1 expressed by
cultured fibroblasts are co-regulated by ECM stiffness. This hypothesis is derived from
the fact that ECM stiffness is the third pivotal factor for myofibroblast differentiation.
Mechanical stress increases ED-A FN expression in wound granulation tissue (Hinz et
al., 2001a). Hence, in my second objective, I aimed to determine whether ECM stiffness
regulates the expression of ED-A FN and LTBP-1 in a conjunct manner. I first tested this
idea with Western blotting of cell lysates from HDFs cultured in the absence and
presence of pro-fibrotic TGFβ-1 on silicone substrates with different stiffness.
41
Figure 9: The effect of ECM stiffness on the expression levels of ED-A FN and LTBP-1.
Substrates with 5 kPa represented normal connective tissue (dermis) whereas 100
kPa was used to mimic the mechanics of fibrotic tissue (Hinz, 2010c). 3,000 kPa
substrates were used to provide conditions close to conventional culture plastic.
Independent of TGF -1, the expression levels of both ED-A FN and LTBP-1 increased
with increasing substrate stiffness. The magnitude of expression is greater and increased
stronger in the presence of TGF -1 (Fig. 9).
Next, I investigated the influence of substrate stiffness on co-expression and
organization of LTBP-1 and ED-A FN by immunofluorescence analysis. In the absence
of TGF -1, on 5 kPa soft substrates, ED-A FN appeared as thin microfibrils, while there
appeared to be very little LTBP-1 organized (Fig. 10). With increasing stiffness, ED-A
FN fibrils were more condensed and thicker, and assembled in a parallel orientation to
the direction of the cells. At 100 kPa, LTBP-1 was visibly becoming more organized into
Western blot analysis was used to support immunofluorescence experiments shown in Fig. 10
and 11. The same antibodies used in immunofluorescence were also used for Western blotting.
GAPDH was used as loading control. The graph (below) depicts the average expression level
evaluated for both ED-A FN (red) and LTBP-1 (green) across increasing ECM stiffness in
conditions with or without TGF -1 (N=3).
42
thin fibres. At a higher stiffness of 3,000 kPa, ED-A FN now appeared to be more highly
organized, dense ECM, while LTBP-1 became more arranged as highly condensed
fibrillar structures (Fig. 11).
Figure 10: The effect of stiffness on ED-A FN and LTBP-1 organization.
In the presence of exogenous TGF -1, the organization and assembly of both ED-A
FN and LTBP-1 followed the same trend as observed without the addition TGF -1.
However, with TGF -1 added, both proteins become much more highly organized and
condensed in their organization as fibrillar structures which could be seen already on the
soft 5 kPa substrate (Fig. 11).
HDFs were seeded on collagen coated differently stiff PDMS substrates for 4d in the absence
of TGF -1. Immunofluorescence was performed for ED-A FN with IST-9 (red) and LTBP-1
with Ab39 (green).
43
Stiffness
ED- A FN LTBP-1 Merge
`100µm
5 k
Pa
10
0 k
Pa
30
00
kP
a
Figure 11: Effect of ECM stiffness and TGFβ-1 on ED-A FN and LTBP-1 organization.
3.3 The 11th
_ED-A_12th
domain in FN appears to be a Binding
Partner of LTBP-1 in the Myofibroblast ECM
So far, I could show that ED-A FN co-immunoprecipates with LTBP-1 in the ECM
of cultured HDFs and that both proteins are co-regulated under mechanical and chemical
in vitro conditions simulating fibrosis. I then set out to test whether the ED-A domain of
cFN could play a specific role in recruiting LTBP-1 to the ECM. For this purpose, I
produced a number of tools to competitively inhibit ED-A FN binding and test binding
capacities of different FN splice variants in solid phase binding assays. The latter were
unfortunately inconclusive at the time of this thesis report and are not presented.
Fig.12 & Fig.13 depict Western blots stained for HIS used to confirm the successful
elution and production of recombinant FN peptides and full FN constructs from both E.
coli BL21 and HEK-293 cells, respectively. For the recombinant FN peptides the 11th
,
ED-A, 11th
_ED-A_12th
, and the 11th
/12th
domains were detected at the expected
HDFs were seeded on collagen coated differently stiff PDMS substrates for 4d this time in the
presence of 2ng/ l TGF -1. Immunofluorescence was performed for ED-A FN with IST-9 (red)
and LTBP-1 with Ab39 (green).
44
molecular weights of 13 kDa, 24 kDa, 52 kDa, and 30 kDa, respectively (Fig.12). Full
FN constructs appeared with molecular weight of 220 kDa as a dimer (Fig.13).
Coomassie blue staining was performed to confirm the purity of the proteins (data not
shown).
Figure 12: Purification of recombinant FN peptides.
Figure 13: Production and Purification of full FN constructs
Full FN constructs were transfected into HEK293 cells and FBS free media were collected. FN constructs are HIS and V5-tagged and were purified by running the medium over a nickel column. Coomassie blue staining (not shown) was used to test for the presence and purity of peptides. Western blot was stained for HIS to confirm the presence of the desired peptides.
Recombinant FN peptides were transfected into E. coli BL21 and induced with IPTG. FN peptides are HIS tagged and purified by running the bacterial lysates over a nickel column. Coomassie blue staining (not shown) was used to test for the presence and purity of peptides. Western blot (shown above) was stained for HIS to detect the desired peptides.
45
It was previously shown that blocking the ED-A domain of FN with specific
antibodies and recombinant ED-A fragments inhibits myofibroblast differentiation (Serini
et al., 1998a). Here, I wanted to test whether these agents have an effect of LTBP-1
incorporation in the myofibroblast ECM. Immunofluorescence results indicated that
incubation with IST-9 affected the incorporation of LTBP-1 into the ECM of HDFs,
while ED-A FN production remained unaffected (Fig.14).
Figure 14: Blocking ED-A with IST-9 affects LTBP-1 incorporation into the ECM.
I then used recombinant FN peptides to act as antagonist to possibly block the
interaction of LTBP-1 with ED-A FN in the HDF ECM. Of all peptides tested, the
11th
_ED-A_12th
peptide had the most dramatic effect on the incorporation of LTBP-1
into the ECM, as seen in immunofluorescence (Fig.15). Control peptides ED-B and 11th
-
12th
domain constructs had no effect on LTBP-1 incorporation.
HDFs were incubated with IST-9 (300 g/ml) for 5d. Controls were incubated in 10% FBS alone
in the absence of IST-9. Immunofluorescence was performed by staining for ED-A FN (IST-9,
red) and LTBP-1 (Ab39, green).
46
Co
ntr
ol
11
th11
th/1
2th
ED- A FN LTBP-1 Merge
19100um
11
th_E
D-A
_12
thE
D-A
100um
Figure 15: The effect of recombinant peptides on LTBP-1 incorporation into the ECM
Similarly to the IST-9 experiment, HDFs were incubated with recombinant FN peptides (200
g/ml) for 5 days. Controls were in 10% FBS medium alone. Immunofluorescence was
performed by staining for ED-A FN (IST-9, red) and LTBP-1 (Ab39, green).
47
Fig.16 displays the Western blot for the recombinant FN peptides with respect to
the total cell lysates. Corresponding to the immunofluorescence results in Fig. 15, that
11th
_ED-A_12th
recombinant peptide had a significant effect on the ability for LTBP-1 to
be incorporated into the ECM.
Figure 16: The effect of recombinant peptides on LTBP-1 incorporation into the ECM
Since there was less LTBP-1 in the ECM, I further analyzed the conditioned
medium to show that there was no affect on the cell’s ability to produce LTBP-1. Rather
cells were unable to incorporate LTBP-1 into the ECM by blocking the interaction with
that specific domain. As shown in Fig. 17, most of the LTBP-1 found in the media
corresponded to HDFs incubated with the 11th
_ED-A_12th
peptide.
HDFs were incubated with recombinant FN peptides (200 g/mL) for 5 days. Controls were in
10% FBS medium alone. Western blotting was performed with HDF and ECM extracts. The
graph (right) depicts the average expression level of LTBP-1 (green) measured in the ECM of
HDFs after incubation with recombinant FN peptides for 5 days (N=3).
48
Figure 17: The effect of recombinant peptides on LTBP-1 secretion into the medium.
Although the IST-9 blocking experiment did suggest that the ED-A domain has a
role in binding to LTBP-1 into the ECM, the effect of the ED-A peptide on LTBP-1 was
not as strong as the 11th
_ED-A_12th
peptide. This suggests that the inclusion of both the
11th
and 12th
domains may enhance the binding of LTBP-1 to the ED-A domain.
3.4 LTBP-1 Expression is Lower in EDA-/-
Mice compared to Wild-type
If ED-A FN plays a role in incorporating and organizing LTBP-1 in the ECM,
fibroblasts deficient for this splice variant should show an appropriate phenotype. To test,
I preformed a number of experiments with ED-A FN KO mice. According to the
immunofluorescence images there was less LTBP-1 organized in the ECM of ED-A-/-
mouse fibroblast compared to wild-type, which was confirmed by the Western blot (Fig.
15). Similar to the recombinant FN peptide experiment, we tested the presence of LTBP-
1 in the medium to confirm that there was no effect on the ability of these cells to
produce LTBP-1. Results showed that most of the LTBP-1, not being incorporated into
the ECM was released into the media instead. Hence, the data from the co-
immunoprecipitation results (Fig. 8) with respect to the media indicated that most of the
HDFs were incubated with recombinant FN peptides (200 g/ml) for 5 days. Controls were in
10% FBS medium alone. Western blotting was performed with conditioned medium.
49
LTBP-1 which is not incorporated into the ECM (total cell lysate) was in fact released
into the medium therefore confirming that these mice have no problem in LTBP-1
production per se.
kDa
250
220
32
LTBP-1
ED-A FN
GAPDH
Total Cell Lysate Media
LTBP-1
kDa
250
Figure 18: LTBP-1 Expression in wild-type and EDA-/- mouse fibroblasts.
Figure 18. LTBP-1 Expression in wild-type and EDA-/-
mouse fibroblasts.
Mouse subcutaneous fibroblasts were seeded for 7 days in culture in FN free 10% FBS medium.
Immunostaining was used to analyze the organization and presence of ED-A FN (red) and
LTBP-1 (green) in the ECM. Western blotting was used to confirm that the ED-A-/-
mice did not
produce ED-A FN. LTBP-1 staining was used to compare the difference in expression in LTBP-
1 in both the total cell lysate and the conditioned medium.
50
Chapter 4: Discussion
Since the discovery of the myofibroblast in the 1970’s by Giulio Gabbiani,
tremendous progress has been made in identifying the factors that lead to its
differentiation. ED-A FN has been well document as an essential FN splice variant
needed for myofibroblast differentiation during wound repair and regeneration (Serini et
al., 1998a; Serini and Gabbiani, 1999a; White et al., 2008). Two other factors, TGF -1
and mechanical stress (ECM stiffness) have also been reported to be crucial for
myofibroblast differentiation (Hinz, 2010c). Cell traction forces exerted to LAP through
an integrin-mediated mechanism, results in a conformation change of the latent complex
that liberates active TGFβ-1(Wipff et al., 2007). The mechanical liberation of this growth
factor is essential in the normal tissue repair by enhancing the inflammatory response
thereby generating a positive feed forward signal that stimulates not only myofibroblast
differentiation, but also the production of ECM, and an increase and decrease in the
synthesis of tissue inhibitors of metalloproteinases and proteases, respectfully (Hinz,
2009). However, whether these two required factors associate or collaborate with ED-A
FN still remains undefined today. The purpose of my thesis was to find a link between
mechanical stress, TGF -1, and ED-A FN. In my thesis, I hypothesized that the role of
ED-A domain of FN plays a role in sequestering the LLC in the ECM, and that
mechanical stress is a regulator in the expression and organization of both ED-A FN and
LTBP-1 in the myofibroblast ECM.
4.1 Discussion
ED-A FN is an intricate factor involved in myofibroblast differentiation (Kohan et
al., 2010; Serini et al., 1998a; White et al., 2008). However, there are still biological
aspects that need to be uncovered in order to fully understand the role of ED-A FN in
normal wound healing and in fibrotic disorders. Many studies have elucidated that ED-A
FN is involved in various integrin-related mediated activities such as cellular adhesion
and cell cycle progression (Manabe et al. 1999). However, my data suggest that, in
particular the 11th
_ED-A_12th
domain of FN may also be involved in storing latent
51
TGF -1 in the ECM by specifically interacting with LTBP-1. Many studies have shown
that FN acts as an essential preliminary scaffold for the proper deposition and assembly
of LTBP-1 into fibrillar networks (Unsold et al., 2001). However, the mechanism of
binding and sequestration of LLC into the ECM and its relevance for the release of active
TGF -1 remain poorly understood. Moreover, it has not been tested whether specific FN
splice variants play specific roles in this process. Cellular FN is the only type of FN
expressed and found in normal connective tissues. ED-A FN is highly expressed and
synthesized by myofibroblasts during wound repair. Although the bulk of the tissue is
plasma derived (Moretti et al., 2007), my data shows that the presence of the 11th
_ED-
A_12th
domain of FN is necessary for LTBP-1 to be incorporated into the myofibroblast
ECM. This suggests that newly synthesized cFN is essential in regulating wound healing.
4.1.1 The role of ECM stiffness in LTBP-1 binding to ED-A FN
Since the background of my work is based on skin wound healing and fibrosis, I
first used co-immunoprecipitation to decipher whether there was an association between
ED-A FN and LTBP-1 in cultured dermal fibroblasts and myofibroblasts. In addition, by
comparing the LTBP-1 expression in both wild-type and EDA-/-
mouse fibroblasts, I
found more LTBP-1 released into the media of EDA-/-
fibroblasts than in wild-type.
LTBP-1 presence in the ECM of wild-type fibroblasts was higher compared to the EDA-/-
mouse fibroblasts. I further tested whether binding mostly occurred in the ECM or in
conditioned culture medium. My results confirmed that most of the ED-A FN did in fact
bind to LTBP-1 in the ECM. This observation led me to determine whether the
mechanical stiffness of the ECM was a common regulator in the organization and
expression levels of both ED-A FN and LTBP-1. To test this idea, HDFs were seeded on
top of soft substrates analogous to the normal skin and compared to HDFs that were
grown on a stiff ECM representing mature fibrotic tissue. Unique to my study, I found
that that both the organization and expression of LTBP-1 and ED-A FN increased with
increasing ECM stiffness; protein expression further increased with the addition of
exogenous TGF -1.
52
The question remains how LTBP-1, ED-A FN and the stiffness of the ECM
collaborate to promote myofibroblast differentiation in disease states such as fibrosis. It
has been published that when myofibroblasts are placed in a mechanically challenged
environment, more active TGF -1 is released from the ECM, thereby promoting a
positive feed forward signal in the production of ED-A FN (Wipff et al., 2007). In light
of this fact, it appears not surprising that in the absence of TGF -1 and on a soft ECM
myofibroblasts do not express and organize as much ED-A FN and LTBP-1.
Concomitantly, normal dermis in vivo contains no myofibroblasts. This absence can be
attributed to several factors: 1) lack of a mechanical challenge of cells through ECM-
transmitted forces (intact ECM is mechano-protective), 2) low (or no) levels of ED-A
FN, which is only upregulated during embryogenesis and wound healing, and 3) low
levels of LTBP-1 microfibrils.
These conditions contribute to sustaining and maintaining tissue homeostasis.
However when tissue injury occurs, the induction of fibroblast migration towards the
wounding site and the synthesis of ED-A FN is initiated by the presence of TGF -1
released by inflammatory cells such as macrophages and neutrophils. It has been shown
with cultured fibroblasts that TGF -1 induces the insertion of both the ED-A and ED-B
domains within FN, and even more so for the ED-A domain (Serini et al., 1998b). ED-A
FN acts as a preliminary scaffold and adhesive substrate for fibroblasts. This attachment
is mediated partly by ED-A-specific integrins such as 4 7, which has been shown to be
expressed in mouse and human lung myofibroblasts (Kohan et al., 2010). Fibroblast
attachment through the ED-A-mediated integrins generates a positive feedback loop of
myofibroblast differentiation. In the continued presence of TGF -1, they begin to
synthesize and secrete their own latent TGF -1, which is required to differentiate into
myofibroblasts and retain this phenotype.
In order for latent TGF -1 to be stored in the ECM, it is secreted as a SLC; TGFβ1
is non-covalently bound to LAP in the SLC that in turn is covalently bound to LTBP-1
via disulphide bridges. In turn, LTBP-1 binds to FN and possibly other proteins in the
ECM (Annes et al., 2003; Isogai et al., 2003; Ramirez and Rifkin, 2009; Todorovic et al.,
53
2005). Colocalization of ED-A FN with LTBP-1 has been demonstrated in the ECM of
cultured rat lung myofibroblast (Wipff et al., 2007). The continuous synthesis of ED-A
FN, now assembling with LTBP-1 leads to a more condensed and higher-ordered ECM.
The outcome of this organization is overall stiffening of the ECM. Stiffening in turn
initiates the activation of latent TGF -1 through an integrin-related mechanism by
pulling on the RGD sequence found in LAP (Wipff et al., 2007) (Buscemi et al., 2011b).
The epithelial integrin αvβ6 was found to be capable of activating latent TGF-β1
both in vitro and in vivo; this action strongly contributed to the promotion of lung fibrosis
(Annes et al., 2003; Jenkins et al., 2006; Munger et al., 1999). Integrin αvβ6 is known to
liberate latent TGFβ-1 by binding to the hinge region of LTBP-1 (Annes et al., 2004)
(Fontana et al., 2005). However, the precise mechanism of how the latent TGFβ-1 is
activated by this integrin remained undefined. Speculation that epithelial cell-mediated
traction has been proposed yet no direct evidence has been provided so far (Annes et al.,
2004; Keski-Oja et al., 2004). The idea of αvβ6-integrin mediated contraction however
remains controversial since the epithelium does become less prominent as fibrosis
progresses (Wipff and Hinz, 2008). Traction activation of TGFβ-1 has been demonstrated
for fibroblasts (Wipff et al., 2007; Zhou et al., 2010) and mechanical forces have been
show to liberate TGFβ-1 from the LLC in blood plasma (Ahamed et al., 2008) and in a
cell free system (Buscemi et al., 2011a). The fibroblast integrin(s) involved in mediating
the binding to the RGD sequence in LAP still remain unknown and are currently studied
in the lab; αvβ5 integrin emerges as our attractive candidate (unpublished).
4.1.2 Specific binding of LTBP-1 to ED-A?
My data shows that ED-A FN is involved in binding to LTBP-1 in the ECM.
However, it remains unclear whether the ED-A domain in FN specifically associates with
the LTBP-1 or whether LTBP-1 has a preferential binding to the ED-A domain. By using
IST-9, which specifically blocks the ED-A domain, I show reduced LTBP-1
incorporation into the ECM. This result strongly suggests that the ED-A domain has a
direct role in storing or recruiting LLC in the ECM. Moreover, addition of the peptides
comprising 11th
_ED-A_12th
domain of FN to HDF cultures strongly antagonize LTBP-1
54
incorporation into the ECM. Using only recombinant ED-A peptides as antagonists had a
lesser inhibitory effect on the level of LTBP-1 incorporation into the ECM. These results
suggests that the 11th
_ED-A_12th
domain, rather than the ED-A domain alone, could play
a substantial role in LTBP-1 binding to FN. Hence, the ED-A domain alone may not be
sufficient enough to bind to LTBP-1, but requires the assistance of neighbouring binding
domains to fully cross-link LTBP-1 into the ECM.
The question is raised how the flanking domains of ED-A could influence the
LTBP-1 binding process. Many studies support the role of heparin as key linkage factor
involved in the incorporation and stabilization of LTBP-1 in the ECM. LTBP-1, which
has a sensitive proline-rich “hinge” region, contains a heparin binding consensus
sequence between amino acids 414-425 located at the N terminus (Chen et al., 2007).
Further, FN contains two heparin-binding domains: one close to the N terminus (Hep1
domain), the other located between the III12-14 domains called the Hep2 domain (Clark
et al., 2003). According to Chen and coworkers, heparin sulphate proteoglycans (HSPGs)
that are found in these heparin-binding domains are critical in regulating the TGF -1
availability by controlling the deposition of LTBP-1 into the ECM (Chen et al., 2007).
Since LTBP-1 belongs to the fibrillin family, and fibrillins are known to depend on
HSPGs for their incorporation into the ECM, possibly the III12-14 domain may aid the
ED-A domain in the stabilization and binding of LTBP-1 to FN. This idea is strongly
supported by the fact that heparin-binding domains in FN were shown to be involved in
regulating the binding of a variety of growth factors, including TGF -1, human growth
factors, CTGF, and PDGF (Kohan et al., 2010).
An indication that heparin plays a strong role in LTBP-1 assembly was given by
incubating fetal rat calvarial cells in the presence of heparin inhibitors (Chen et al., 2007).
The authors found that in the presence of heparin inhibitors there was a lower level of
LLC incorporated into the ECM, and more LTBP-1 found in the media. Since there are
two heparin binding domains in FN, Chen and coworkers used single binding assay to
rule out that the Hep1 domain is not critical in mediating the linkage between LTBP-1
and FN. These results suggest, although this has never been tested, that Hep2 domain
may be the possible candidate. Thus, the inclusion of the Hep2 domain may be the reason
55
why blocking the ED-A domain alone with IST-9 is not sufficient to completely hinder
the incorporation of LTBP-1 into the ECM, in contrast to the full action of the
recombinant 11th
_ED-A_12th
peptide. Overall these results strongly support the fact that
although the ED-A domain may have a role in binding to LTBP-1, alone it may not be
enough to fully incorporate LTBP-1 into the ECM. I speculate that LTBP-1 binding
requires the assistance of neighbouring linkage partners such as the presence of the Hep2
binding domain which is needed for LTBP-1 assembly and adherence to the ECM.
Another possibility is that incorporation of the ED-A domain in FN reveals cryptic
sites for the binding of ED-A FN binding integrins, such as integrin 4 7 and 4 1.
Both integrins are involved in mediating TGF -1 related biological activities, such as
cellular adhesion, differentiation, and migration (White et al., 2008) The incorporation of
ED-A domain which is known to enhance fibroblast adhesion and stabilization to the
ECM, may also assist in storing LTBP-1 by up-regulating specific integrins that bind to
the RGD sequence present in the LTBP-1 protein.
4.1.3 Crosslinking of LTBP-1 with the ED-A FN ECM
The crosslinking of LTBP-1 is just as essential as the presence of a stiff ECM in the
differentiation of myofibroblast. The low percentage of -SMA positive myofibroblasts
in a compliant ECM is partly due to the fact that fibroblasts fail to develop enough
contractility to release latent TGF -1 from LAP (Wipff et al., 2007). Similarly, LTBP-1
that is secreted but not bound to the ECM would have that same effect (Annes et al.,
2004). Integrins could still recognize the RGD sequence on LTBP-1; however they would
fail to release the latent TGF -1 since the high contraction associated with pulling on the
LAP may separate the whole LLC from the ECM. These ideas correspond well with a
study from Muro and coworkers in which they showed that the ED-A-/-
lung
myofibroblasts retained the ability to synthesize and secrete latent TGF -1. However
their ability to activate latent TGF -1 from the ECM was significantly hindered
compared to their wild-type counterparts (Muro et al., 2008). In my experiments, the lack
of sufficient cross-linking of the LTBP-1 to the ECM could explain why most of the
LTBP-1 was found in the conditioned medium, in conditions where HDFs were
56
incubated with the recombinant 11th
_ED-A_12th
FN peptide rather just the ED-A peptide
alone.
4.1.4 The role of ED-A FN binding integrins and alternative factors
Currently integrin α4β1 is only known to be expressed in cultured dermal fibroblasts (i.e.
in vitro), but not in vivo. Gailit and coworkers suggested that α4β1 may normally be
absent or present at very low concentrations in dermal fibroblasts; it is up-regulated in the
presence of cytokines during wound healing or in inflammatory skin diseases (Gailit et
al., 1993). This situation however has not been investigated. The signalling pathways
initiated as a result of ED-A FN interaction with α4β1, and the possible connection to
TGFβ-1 signalling and TGFβ-1-induced α-SMA expression remains elusive.
Furthermore, the specific factors involved in regulating ED-A FN expression are
currently undefined. Recent evidence by White and coworkers suggests possible
regulation by a tumour suppressor gene, known as phosphatase and tensin homolog
deleted on chromosome 10 or PTEN, where an inverse relationship between ED-A FN
and PTEN levels was identified in IPF affected patients (White et al., 2006; White et al.,
Figure 19: Schematic of the PI3K/Akt/mTOR axis involved in the regulation of ED-A FN
alternative splicing and fibroblast activity.
mTOR form a complex with rictor and gβL which leads to the initial phosphorylation of S473
on Akt, then subsequent phosphorylation of T308
by PDK1. PTEN is a negative regulator of
this activity. Activation of Akt indirectly initiates ED-A exon splicing by phosphorylating and
activating the splicing factor, SF2/ASF. FN translation is enhanced by the mTOR/raptor/gβL
complex (White et al., 2010).
57
2008). PTEN, which was found to be loss or inhibited in lung fibroblasts isolated from
IPF patients, is known to be involved in several biological activities including: inhibiting
cell migration and growth, and promoting cellular apoptosis (White et al., 2006).
Interestingly, TGF which is a potent inducer of ED-A exon splicing and a negative
regulator of PTEN was found to enhance the PI3K/Akt signalling pathway; a pathway
involved in the biological activation of fibroblasts, including proliferation and collagen
synthesis (Conte et al., 2011). Furthermore, it is the loss of PTEN activity that allows for
myofibroblast differentiation and fibroblast migration to occur. In addition, the
activation of Akt due to the phosphorylation by the mammalian target of Rapamycin
(mTOR)/rictor/g L complex, is an indirect regulator involved in ED-A exon splicing by
enhancing the activity of the SF2/ASF splicing factor. Thus, experiments have shown that
in the absence of PTEN, the increase in Akt activity not only enhances FN production but
a greater percentage of the FN molecules contain the ED-A domain. These ideas generate
a strong connection between TGF and PTEN and the regulation of ED-A FN alternative
splicing and translation, and can be summed up in molecular detail as shown in Fig.19.
The suppression of PTEN expression by the presence of TGF stimulates
PI3K/Akt/mTOR signalling cascade which overall enhances the synthesis of ED-A
splicing, and subsequent induction of fibroblast activation and proliferation. Overall, the
study proposed by White et al., 2010 suggests that the PI3K/Akt/mTOR pathway could
be a potential therapeutic target in limiting the excessive ECM production that is well
known in the generation and sustenance of fibrosis (White et al., 2010).
4.2 Conclusion - ED-A FN as a Therapeutic Anti-Fibrosis Target?
In summary, our data suggest that the ED-A domain in FN plays a role in the
association with LTBP-1 in the myofibroblast ECM. The full stabilization of LTBP-1
into the ECM, however, requires the possible assistance of neighbouring HSPGs located
in the Hep2 domain. Strong cross-linking of LTBP-1 is needed to allow release of latent
TGF -1 during wounding, ultimately leading to myofibroblast differentiation. In this
process, the increase in ECM stiffness in conjunction with the up-regulation of specific
integrins associated with the presence of the ED-A domain, seem to be required. These
58
findings provide a clearer picture and more in depth look on how these three essential
factors are linked, which will be essential to develop therapeutic treatments that target
specific factors associated with the deregulation myofibroblast activity during fibrosis.
We still have to better understand the role of ED-A FN regulation in myofibroblast
differentiation. Nevertheless, the current knowledge on ED-A FN deregulation in the
development of fibroproliferative and contractive disorders provides an advance step in
steering the development of possible therapeutic treatments that target ED-A FN (Fig.20).
Blocking the expression of ED-A FN can be a potential target for therapy for several
reasons: a) it is an ECM molecule, and therefore easily accessible b) it is found normally
in low levels in adults, hence very few potential side effects as a result of blocking the
ED-A domain directly, or blocking the downstream molecules that regulate ED-A
alternative splicing c) it is only synthesized by myofibroblasts in all pathological and
physiological (wound healing) settings: Dupuytren’s disease, organ fibrosis, arterial
intimal thickening, and stroma reaction to epithelial cancers (Serini et al., 1998b)
59
Figure 20: Potential therapeutic strategies targeting the differentiation of myofibroblast.
TGFβ-1 was also suggested as a potential target to reduce the up-regulation of ED-
A FN; however since TGFβ-1 is also involved in generating an innate immune response,
the risk of opportunistic infections and development of autoimmune-like diseases led to
disregard blocking TGFβ-1 as a potential option for therapy (Serini and Gabbiani, 1999b)
4.3 Outlook
My future plans include doing cryosectioning and comparing the LTBP-1
expression in the dermal tissue of wild type mice compared to EDA-/-
mice. Since it has
been recently shown that total FN-/-
fibroblasts fail to incorporate LTBP-1 into the ECM,
I plan to do a rescue experiment. This experiment involves seeding total FN-/-
fibroblasts
onto the different full FN constructs and recombinant FN peptides to determine which
Different chemical and mechanical factors can be used to target components that are essential to
myofibroblast differentiation. This includes using antibodies and recombinant peptides that
target the ED-A domain such as IST-9 and recombinant ED-A peptides, respectively, or
targeting pathways directly such as the PI3K/Akt/mTOR signalling cascade as suggested by
White et al., 2010. Other inhibitors include targeting the pro-fibrotic cytokine TGFβ-1, however
this is not an optimal choice since TGFβ-1 is involved in mediating the immune system as well
(Reproduced from (Hinz and Gabbiani, 2010a)).
60
specific FN construct and in which specific domain of the FN can rescue LTBP-1
incorporation into the ECM. In addition, the same experiment would be repeated under
stretch, since it is known that stretching FN reveals cryptic sites that might enhance the
binding of LTBP-1 into the matrix. Finally, I plan to do the solid phase binding assay to
determine if the binding between LTBP-1 and the full FN constructs and recombinant FN
peptide occurs by direct binding, and similar to the rescue experiment above, to see
which specific FN variant and domain is involved in LTBP-1 binding.
61
References:
Ahamed, J., N. Burg, K. Yoshinaga, C.A. Janczak, D.B. Rifkin, and B.S. Coller. 2008. In vitro and in vivo evidence for shear-induced activation of latent transforming growth factor-beta1. Blood. 112:3650-3660.
Amatangelo, M.D., D.E. Bassi, A.J. Klein-Szanto, and E. Cukierman. 2005. Stroma-derived three-dimensional matrices are necessary and sufficient to promote desmoplastic differentiation of normal fibroblasts. Am J Pathol. 167:475-488.
Annes, J.P., Y. Chen, J.S. Munger, and D.B. Rifkin. 2004. Integrin {alpha}V{beta}6-mediated activation of latent TGF-{beta} requires the latent TGF-{beta} binding protein-1. J Cell Biol. 165:723-734.
Annes, J.P., J.S. Munger, and D.B. Rifkin. 2003. Making sense of latent TGFbeta activation. J Cell Sci. 116:217-224.
Aplin, A.E., A. Howe, S.K. Alahari, and R.L. Juliano. 1998. Signal transduction and signal modulation by cell adhesion receptors: the role of integrins, cadherins, immunoglobulin-cell adhesion molecules, and selectins. Pharmacol Rev. 50:197-263.
Aplin, A.E., A.K. Howe, and R.L. Juliano. 1999. Cell adhesion molecules, signal transduction and cell growth. Curr Opin Cell Biol. 11:737-744.
Arora, P.D., and C.A.G. Mcculloch. 1994. Dependence of Collagen Remodeling on Alpha-Smooth Muscle Actin Expression by Fibroblasts. J Dent Res. 73:209-209.
Arthur, M.J. 2000. Fibrogenesis II. Metalloproteinases and their inhibitors in liver fibrosis. Am J Physiol Gastrointest Liver Physiol. 279:G245-249.
Atfi, A., K. Lepage, P. Allard, A. Chapdelaine, and S. Chevalier. 1995. Activation of a serine/threonine kinase signaling pathway by transforming growth factor type beta. Proc Natl Acad Sci U S A. 92:12110-12114.
Bae, E., T. Sakai, and D.F. Mosher. 2004. Assembly of exogenous fibronectin by fibronectin-null cells is dependent on the adhesive substrate. J Biol Chem. 279:35749-35759.
Balamayooran, G., S. Batra, T. Balamayooran, S. Cai, and S. Jeyaseelan. 2011. Monocyte chemoattractant protein 1 regulates pulmonary host defense via neutrophil recruitment during Escherichia coli infection. Infect Immun. 79:2567-2577.
Bayat, A., D.A. McGrouther, and M.W. Ferguson. 2003. Skin scarring. BMJ. 326:88-92. Benecky, M.J., C.G. Kolvenbach, R.W. Wine, J.P. DiOrio, and M.W. Mosesson. 1990.
Human plasma fibronectin structure probed by steady-state fluorescence polarization: evidence for a rigid oblate structure. Biochemistry. 29:3082-3091.
Birkedal-Hansen, H., W.G. Moore, M.K. Bodden, L.J. Windsor, B. Birkedal-Hansen, A. DeCarlo, and J.A. Engler. 1993. Matrix metalloproteinases: a review. Crit Rev Oral Biol Med. 4:197-250.
Border, W.A., S. Okuda, L.R. Languino, M.B. Sporn, and E. Ruoslahti. 1990. Suppression of experimental glomerulonephritis by antiserum against transforming growth factor beta 1. Nature. 346:371-374.
62
Brakebusch, C., and R. Fassler. 2003. The integrin-actin connection, an eternal love affair. EMBO J. 22:2324-2333.
Bucala, R., L.A. Spiegel, J. Chesney, M. Hogan, and A. Cerami. 1994. Circulating fibrocytes define a new leukocyte subpopulation that mediates tissue repair. Mol Med. 1:71-81.
Buscemi, L., D. Ramonet, F. Klingberg, A. Formey, J. Smith-Clerc, J.-J. Meister, and B. Hinz. 2011a. The single-molecule mechanics of the latent TGF 1 complex. Current Biology. in press.
Buscemi, L., D. Ramonet, F. Klingberg, A. Formey, J. Smith-Clerc, J.J. Meister, and B. Hinz. 2011b. The Single-Molecule Mechanics of the Latent TGF-beta1 Complex. Curr Biol. 21:2046-2054.
Butcher, D.T., T. Alliston, and V.M. Weaver. 2009. A tense situation: forcing tumour progression. Nat Rev Cancer. 9:108-122.
Campaner, A.B., L.M. Ferreira, A. Gragnani, J.M. Bruder, J.L. Cusick, and J.R. Morgan. 2006. Upregulation of TGF-beta1 expression may be necessary but is not sufficient for excessive scarring. J Invest Dermatol. 126:1168-1176.
Carter, W.G., E.A. Wayner, T.S. Bouchard, and P. Kaur. 1990. The role of integrins alpha 2 beta 1 and alpha 3 beta 1 in cell-cell and cell-substrate adhesion of human epidermal cells. J Cell Biol. 110:1387-1404.
Castelletti, F., R. Donadelli, F. Banterla, F. Hildebrandt, P.F. Zipfel, E. Bresin, E. Otto, C. Skerka, A. Renieri, M. Todeschini, J. Caprioli, R.M. Caruso, R. Artuso, G. Remuzzi, and M. Noris. 2008. Mutations in FN1 cause glomerulopathy with fibronectin deposits. Proc Natl Acad Sci U S A. 105:2538-2543.
Charbonneau, N.L., E.J. Carlson, S. Tufa, G. Sengle, E.C. Manalo, V.M. Carlberg, F. Ramirez, D.R. Keene, and L.Y. Sakai. 2010. In vivo studies of mutant fibrillin-1 microfibrils. J Biol Chem. 285:24943-24955.
Chen, Q., P. Sivakumar, C. Barley, D.M. Peters, R.R. Gomes, M.C. Farach-Carson, and S.L. Dallas. 2007. Potential role for heparan sulfate proteoglycans in regulation of transforming growth factor-beta (TGF-beta) by modulating assembly of latent TGF-beta-binding protein-1. J Biol Chem. 282:26418-26430.
Clark, R.A., J.Q. An, D. Greiling, A. Khan, and J.E. Schwarzbauer. 2003. Fibroblast migration on fibronectin requires three distinct functional domains. J Invest Dermatol. 121:695-705.
Clark, R.A., H.J. Winn, H.F. Dvorak, and R.B. Colvin. 1983. Fibronectin beneath reepithelializing epidermis in vivo: sources and significance. J Invest Dermatol. 80:26s-30s.
Cochrane, C.G. 1978. Mediating systems in inflammatory disease. J Invest Dermatol. 71:40-48.
Conte, E., M. Fruciano, E. Fagone, E. Gili, F. Caraci, M. Iemmolo, N. Crimi, and C. Vancheri. 2011. Inhibition of PI3K prevents the proliferation and differentiation of human lung fibroblasts into myofibroblasts: the role of class I P110 isoforms. PLoS One. 6:e24663.
Dallas, S.L., D.R. Keene, S.P. Bruder, J. Saharinen, L.Y. Sakai, G.R. Mundy, and L.F. Bonewald. 2000. Role of the latent transforming growth factor beta binding
63
protein 1 in fibrillin-containing microfibrils in bone cells in vitro and in vivo. J Bone Miner Res. 15:68-81.
Dallas, S.L., P. Sivakumar, C.J. Jones, Q. Chen, D.M. Peters, D.F. Mosher, M.J. Humphries, and C.M. Kielty. 2005. Fibronectin regulates latent transforming growth factor-beta (TGF beta) by controlling matrix assembly of latent TGF beta-binding protein-1. J Biol Chem. 280:18871-18880.
Dallon, J.C., and H.P. Ehrlich. 2010. Differences in the mechanism of collagen lattice contraction by myofibroblasts and smooth muscle cells. J Cell Biochem. 111:362-369.
Danen, E.H., and K.M. Yamada. 2001. Fibronectin, integrins, and growth control. J Cell Physiol. 189:1-13.
Darby, I.A., T. Bisucci, T.D. Hewitson, and D.G. MacLellan. 1997. Apoptosis is increased in a model of diabetes-impaired wound healing in genetically diabetic mice. Int J Biochem Cell Biol. 29:191-200.
Darby, I.A., T. Bisucci, B. Pittet, S. Garbin, G. Gabbiani, and A. Desmouliere. 2002. Skin flap-induced regression of granulation tissue correlates with reduced growth factor and increased metalloproteinase expression. J Pathol. 197:117-127.
Darby, I.A., and T.D. Hewitson. 2007. Fibroblast differentiation in wound healing and fibrosis. Int Rev Cytol. 257:143-179.
Desmouliere, A., I.A. Darby, and G. Gabbiani. 2003. Normal and pathologic soft tissue remodeling: role of the myofibroblast, with special emphasis on liver and kidney fibrosis. Lab Invest. 83:1689-1707.
Desmoulière, A., A. Geinoz, F. Gabbiani, and G. Gabbiani. 1993. Transforming growth factor-beta 1 induces alpha-smooth muscle actin expression in granulation tissue myofibroblasts and in quiescent and growing cultured fibroblasts. J Cell Biol. 122:103-111.
Ding, Q., C.L. Gladson, H. Wu, H. Hayasaka, and M.A. Olman. 2008. Focal adhesion kinase (FAK)-related non-kinase inhibits myofibroblast differentiation through differential MAPK activation in a FAK-dependent manner. J Biol Chem. 283:26839-26849.
DiPietro, L.A. 1995. Wound healing: the role of the macrophage and other immune cells. Shock. 4:233-240.
Eming, S.A., T. Krieg, and J.M. Davidson. 2007. Inflammation in wound repair: molecular and cellular mechanisms. J Invest Dermatol. 127:514-525.
Engelhardt, E., A. Toksoy, M. Goebeler, S. Debus, E.B. Brocker, and R. Gillitzer. 1998. Chemokines IL-8, GROalpha, MCP-1, IP-10, and Mig are sequentially and differentially expressed during phase-specific infiltration of leukocyte subsets in human wound healing. Am J Pathol. 153:1849-1860.
Erickson, H.P., and N.A. Carrell. 1983. Fibronectin in extended and compact conformations. Electron microscopy and sedimentation analysis. J Biol Chem. 258:14539-14544.
ffrench-Constant, C. 1995. Alternative splicing of fibronectin--many different proteins but few different functions. Exp Cell Res. 221:261-271.
Ffrench-Constant, C., and R.O. Hynes. 1988. Patterns of fibronectin gene expression and splicing during cell migration in chicken embryos. Development. 104:369-382.
64
Fishel, R.S., A. Barbul, W.E. Beschorner, H.L. Wasserkrug, and G. Efron. 1987. Lymphocyte participation in wound healing. Morphologic assessment using monoclonal antibodies. Ann Surg. 206:25-29.
Fontana, L., Y. Chen, P. Prijatelj, T. Sakai, R. Fassler, L.Y. Sakai, and D.B. Rifkin. 2005. Fibronectin is required for integrin alphavbeta6-mediated activation of latent TGF-beta complexes containing LTBP-1. FASEB J. 19:1798-1808.
Fujiwara, N., and K. Kobayashi. 2005. Macrophages in inflammation. Curr Drug Targets Inflamm Allergy. 4:281-286.
Gabbiani, G. 1981. The myofibroblast: a key cell for wound healing and fibrocontractive diseases. Prog Clin Biol Res. 54:183-194.
Gabbiani, G. 1984. [The myofibroblast. A key cell in wound healing and in fibro- contractive diseases of the connective tissue]. Schweiz Rundsch Med Prax. 73:939-941.
Gabbiani, G. 2003. The myofibroblast in wound healing and fibrocontractive diseases. J Pathol. 200:500-503.
Gabbiani, G., G.B. Ryan, and G. Majno. 1971. Presence of modified fibroblasts in granulation tissue and their possible role in wound contraction. Experientia. 27:549-550.
Gailit, J., M. Pierschbacher, and R.A. Clark. 1993. Expression of functional alpha 4 beta 1 integrin by human dermal fibroblasts. J Invest Dermatol. 100:323-328.
George, E.L., E.N. Georges-Labouesse, R.S. Patel-King, H. Rayburn, and R.O. Hynes. 1993. Defects in mesoderm, neural tube and vascular development in mouse embryos lacking fibronectin. Development. 119:1079-1091.
Gibson, M.A., G. Hatzinikolas, E.C. Davis, E. Baker, G.R. Sutherland, and R.P. Mecham. 1995. Bovine latent transforming growth factor beta 1-binding protein 2: molecular cloning, identification of tissue isoforms, and immunolocalization to elastin-associated microfibrils. Mol Cell Biol. 15:6932-6942.
Giltay, R., G. Kostka, and R. Timpl. 1997. Sequence and expression of a novel member (LTBP-4) of the family of latent transforming growth factor-beta binding proteins. FEBS Lett. 411:164-168.
Glukhova, M.A., M.G. Frid, B.V. Shekhonin, T.D. Vasilevskaya, J. Grunwald, M. Saginati, and V.E. Koteliansky. 1989. Expression of extra domain A fibronectin sequence in vascular smooth muscle cells is phenotype dependent. J Cell Biol. 109:357-366.
Goffin, J.M., P. Pittet, G. Csucs, J.W. Lussi, J.J. Meister, and B. Hinz. 2006. Focal adhesion size controls tension-dependent recruitment of alpha-smooth muscle actin to stress fibers. J Cell Biol. 172:259-268.
Grainger, D.J. 2007. TGF-beta and atherosclerosis in man. Cardiovasc Res. 74:213-222.
Gu, L., Y.J. Zhu, X. Yang, Z.J. Guo, W.B. Xu, and X.L. Tian. 2007. Effect of TGF-beta/Smad signaling pathway on lung myofibroblast differentiation. Acta Pharmacol Sin. 28:382-391.
Guan, J.L., J.E. Trevithick, and R.O. Hynes. 1990. Retroviral expression of alternatively spliced forms of rat fibronectin. J Cell Biol. 110:833-847.
Guo, S., and L.A. Dipietro. 2010. Factors affecting wound healing. J Dent Res. 89:219-229.
65
Halliday, N.L., G.M. Rayan, L. Zardi, and J.J. Tomasek. 1994. Distribution of ED-A and ED-B containing fibronectin isoforms in Dupuytren's disease. J Hand Surg Am. 19:428-434.
Hinz, B. 2006. Masters and servants of the force: the role of matrix adhesions in myofibroblast force perception and transmission. Eur J Cell Biol. 85:175-181.
Hinz, B. 2007. Formation and function of the myofibroblast during tissue repair. J Invest Dermatol. 127:526-537.
Hinz, B. 2009. Tissue stiffness, latent TGF-beta1 activation, and mechanical signal transduction: implications for the pathogenesis and treatment of fibrosis. Curr Rheumatol Rep. 11:120-126.
Hinz, B. 2010a. The myofibroblast in connective tissue repair and regeneration. In Regenerative medicine and biomaterials for the repair of connective tissues. C.A.J. Ralphs, editor. Woodhead Publishing Ltd., Cambridge, UK. 39-82.
Hinz, B. 2010b. The myofibroblast: paradigm for a mechanically active cell. J Biomech. 43:146-155.
Hinz, B. 2010c. The myofibroblast: Paradigm for a mechanically active cell. J Biomech. 43:146-155.
Hinz, B., G. Celetta, J.J. Tomasek, G. Gabbiani, and C. Chaponnier. 2001a. Alpha-smooth muscle actin expression upregulates fibroblast contractile activity. Mol Biol Cell. 12:2730-2741.
Hinz, B., and G. Gabbiani. 2010a. Fibrosis: recent advances in myofibroblast biology and new therapeutic perspectives. F1000 Biology Reports. 2:78-82.
Hinz, B., and G. Gabbiani. 2011. The role of myofibroblasts in Dupuytren’s disease: Fundamental aspects of contraction and therapeutic perspectives. In Morbus Dupuytren and Related Hyperproliferative Disorders: Principles, Research, and Clinical Perspectives. M.H.S. C. Eaton, editor. Springer Verlag.
Hinz, B., D. Mastrangelo, C.E. Iselin, C. Chaponnier, and G. Gabbiani. 2001b. Mechanical tension controls granulation tissue contractile activity and myofibroblast differentiation. Am J Pathol. 159:1009-1020.
Hinz, B., S.H. Phan, V.J. Thannickal, A. Galli, M.L. Bochaton-Piallat, and G. Gabbiani. 2007. The myofibroblast: one function, multiple origins. Am J Pathol. 170:1807-1816.
Hinz, B., P. Pittet, J. Smith-Clerc, C. Chaponnier, and J.J. Meister. 2004. Myofibroblast development is characterized by specific cell-cell adherens junctions. Mol Biol Cell. 15:4310-4320.
Hu, B., Z. Wu, and S.H. Phan. 2003. Smad3 mediates transforming growth factor-beta-induced alpha-smooth muscle actin expression. Am J Respir Cell Mol Biol. 29:397-404.
Hu, Y., J. Peng, D. Feng, L. Chu, X. Li, Z. Jin, Z. Lin, and Q. Zeng. 2006. Role of extracellular signal-regulated kinase, p38 kinase, and activator protein-1 in transforming growth factor-beta1-induced alpha smooth muscle actin expression in human fetal lung fibroblasts in vitro. Lung. 184:33-42.
Humphries, J.D., A. Byron, and M.J. Humphries. 2006. Integrin ligands at a glance. J Cell Sci. 119:3901-3903.
66
Ishida, Y., T. Kondo, T. Takayasu, Y. Iwakura, and N. Mukaida. 2004. The essential involvement of cross-talk between IFN-gamma and TGF-beta in the skin wound-healing process. J Immunol. 172:1848-1855.
Isogai, Z., R.N. Ono, S. Ushiro, D.R. Keene, Y. Chen, R. Mazzieri, N.L. Charbonneau, D.P. Reinhardt, D.B. Rifkin, and L.Y. Sakai. 2003. Latent transforming growth factor beta-binding protein 1 interacts with fibrillin and is a microfibril-associated protein. J Biol Chem. 278:2750-2757.
Janmey, P.A., P.C. Georges, and S. Hvidt. 2007. Basic rheology for biologists. Methods Cell Biol. 83:3-27.
Janmey, P.A., J.P. Winer, M.E. Murray, and Q. Wen. 2009. The hard life of soft cells. Cell Motil Cytoskeleton. 66:597-605.
Jarnagin, W.R., D.C. Rockey, V.E. Koteliansky, S.S. Wang, and D.M. Bissell. 1994. Expression of variant fibronectins in wound healing: cellular source and biological activity of the EIIIA segment in rat hepatic fibrogenesis. J Cell Biol. 127:2037-2048.
Jenkins, G. 2008. The role of proteases in transforming growth factor-beta activation. Int J Biochem Cell Biol. 40:1068-1078.
Jenkins, R.G., X. Su, G. Su, C.J. Scotton, E. Camerer, G.J. Laurent, G.E. Davis, R.C. Chambers, M.A. Matthay, and D. Sheppard. 2006. Ligation of protease-activated receptor 1 enhances alpha(v)beta6 integrin-dependent TGF-beta activation and promotes acute lung injury. J Clin Invest. 116:1606-1614.
Kanzaki, T., A. Olofsson, A. Moren, C. Wernstedt, U. Hellman, K. Miyazono, L. Claesson-Welsh, and C.H. Heldin. 1990. TGF-beta 1 binding protein: a component of the large latent complex of TGF-beta 1 with multiple repeat sequences. Cell. 61:1051-1061.
Kaplan, M.J. 2011. Neutrophils in the pathogenesis and manifestations of SLE. Nat Rev Rheumatol. 7:691-699.
Keski-Oja, J., K. Koli, and H. von Melchner. 2004. TGF-beta activation by traction? Trends Cell Biol. 14:657-659.
Kinsey, R., M.R. Williamson, S. Chaudhry, K.T. Mellody, A. McGovern, S. Takahashi, C.A. Shuttleworth, and C.M. Kielty. 2008. Fibrillin-1 microfibril deposition is dependent on fibronectin assembly. J Cell Sci. 121:2696-2704.
Kirk, T.Z., M.E. Mark, C.C. Chua, B.H. Chua, and M.D. Mayes. 1995. Myofibroblasts from scleroderma skin synthesize elevated levels of collagen and tissue inhibitor of metalloproteinase (TIMP-1) with two forms of TIMP-1. J Biol Chem. 270:3423-3428.
Kirsner, R.S., and W.H. Eaglstein. 1993. The wound healing process. Dermatol Clin. 11:629-640.
Kisseleva, T., and D.A. Brenner. 2011. Is it the end of the line for the EMT? Hepatology. 53:1433-1435.
Kjalke, M., A. Silveira, A. Hamsten, U. Hedner, and M. Ezban. 2000. Plasma lipoproteins enhance tissue factor-independent factor VII activation. Arterioscler Thromb Vasc Biol. 20:1835-1841.
Kohan, M., A.F. Muro, E.S. White, and N. Berkman. 2010. EDA-containing cellular fibronectin induces fibroblast differentiation through binding to alpha4beta7
67
integrin receptor and MAPK/Erk 1/2-dependent signaling. FASEB J. 24:4503-4512.
Koli, K., M. Hyytiainen, M.J. Ryynanen, and J. Keski-Oja. 2005. Sequential deposition of latent TGF-beta binding proteins (LTBPs) during formation of the extracellular matrix in human lung fibroblasts. Exp Cell Res. 310:370-382.
Koli, K., M.J. Ryynanen, and J. Keski-Oja. 2008. Latent TGF-beta binding proteins (LTBPs)-1 and -3 coordinate proliferation and osteogenic differentiation of human mesenchymal stem cells. Bone. 43:679-688.
Konter, U., I. Kellner, E. Klein, R. Kaufmann, V. Mielke, and W. Sterry. 1989. Adhesion molecule mapping in normal human skin. Arch Dermatol Res. 281:454-462.
Kornblihtt, A.R., C.G. Pesce, C.R. Alonso, P. Cramer, A. Srebrow, S. Werbajh, and A.F. Muro. 1996. The fibronectin gene as a model for splicing and transcription studies. FASEB J. 10:248-257.
Kornblihtt, A.R., K. Vibe-Pedersen, and F.E. Baralle. 1984. Human fibronectin: cell specific alternative mRNA splicing generates polypeptide chains differing in the number of internal repeats. Nucleic Acids Res. 12:5853-5868.
Koski, C., J. Saharinen, and J. Keski-Oja. 1999. Independent promoters regulate the expression of two amino terminally distinct forms of latent transforming growth factor-beta binding protein-1 (LTBP-1) in a cell type-specific manner. J Biol Chem. 274:32619-32630.
Kuhn, C., 3rd, J. Boldt, T.E. King, Jr., E. Crouch, T. Vartio, and J.A. McDonald. 1989. An immunohistochemical study of architectural remodeling and connective tissue synthesis in pulmonary fibrosis. Am Rev Respir Dis. 140:1693-1703.
Laping, N.J., E. Grygielko, A. Mathur, S. Butter, J. Bomberger, C. Tweed, W. Martin, J. Fornwald, R. Lehr, J. Harling, L. Gaster, J.F. Callahan, and B.A. Olson. 2002. Inhibition of transforming growth factor (TGF)-beta1-induced extracellular matrix with a novel inhibitor of the TGF-beta type I receptor kinase activity: SB-431542. Mol Pharmacol. 62:58-64.
Larjava, H., C. Wiebe, C. Gallant-Behm, D.A. Hart, J. Heino, and L. Hakkinen. 2011. Exploring scarless healing of oral soft tissues. J Can Dent Assoc. 77:b18.
Lavigueur, A., H. La Branche, A.R. Kornblihtt, and B. Chabot. 1993. A splicing enhancer in the human fibronectin alternate ED1 exon interacts with SR proteins and stimulates U2 snRNP binding. Genes Dev. 7:2405-2417.
Leask, A. 2007. TGFbeta, cardiac fibroblasts, and the fibrotic response. Cardiovasc Res. 74:207-212.
Leask, A., and D.J. Abraham. 2004. TGF-beta signaling and the fibrotic response. Faseb J. 18:816-827.
Leask, A., S.K. Parapuram, X. Shi-Wen, and D.J. Abraham. 2009. Connective tissue growth factor (CTGF, CCN2) gene regulation: a potent clinical bio-marker of fibroproliferative disease? J Cell Commun Signal. 3:89-94.
Lee, J.M., S. Dedhar, R. Kalluri, and E.W. Thompson. 2006. The epithelial-mesenchymal transition: new insights in signaling, development, and disease. J Cell Biol. 172:973-981.
Lowrie, A.G., D.M. Salter, and J.A. Ross. 2004. Latent effects of fibronectin, alpha5beta1 integrin, alphaVbeta5 integrin and the cytoskeleton regulate pancreatic carcinoma cell IL-8 secretion. Br J Cancer. 91:1327-1334.
68
Magnusson, M.K., and D.F. Mosher. 1998. Fibronectin: structure, assembly, and cardiovascular implications. Arterioscler Thromb Vasc Biol. 18:1363-1370.
Manabe, R., N. Oh-e, and K. Sekiguchi. 1999. Alternatively spliced EDA segment regulates fibronectin-dependent cell cycle progression and mitogenic signal transduction. J Biol Chem. 274:5919-5924.
Manabe, R., N. Ohe, T. Maeda, T. Fukuda, and K. Sekiguchi. 1997. Modulation of cell-adhesive activity of fibronectin by the alternatively spliced EDA segment. J Cell Biol. 139:295-307.
Mardon, H.J., G. Sebastio, and F.E. Baralle. 1987. A role for exon sequences in alternative splicing of the human fibronectin gene. Nucleic Acids Res. 15:7725-7733.
Massam-Wu, T., M. Chiu, R. Choudhury, S.S. Chaudhry, A.K. Baldwin, A. McGovern, C. Baldock, C.A. Shuttleworth, and C.M. Kielty. 2010. Assembly of fibrillin microfibrils governs extracellular deposition of latent TGF beta. J Cell Sci. 123:3006-3018.
McAnulty, R.J. 2007. Fibroblasts and myofibroblasts: their source, function and role in disease. Int J Biochem Cell Biol. 39:666-671.
Meek, R.M., S. McLellan, and J.F. Crossan. 1999. Dupuytren's disease. A model for the mechanism of fibrosis and its modulation by steroids. J Bone Joint Surg Br. 81:732-738.
Midwood, K.S., L.V. Williams, and J.E. Schwarzbauer. 2004. Tissue repair and the dynamics of the extracellular matrix. Int J Biochem Cell Biol. 36:1031-1037.
Moir, L.M., J.K. Burgess, and J.L. Black. 2008. Transforming growth factor beta 1 increases fibronectin deposition through integrin receptor alpha 5 beta 1 on human airway smooth muscle. J Allergy Clin Immunol. 121:1034-1039 e1034.
Moren, A., A. Olofsson, G. Stenman, P. Sahlin, T. Kanzaki, L. Claesson-Welsh, P. ten Dijke, K. Miyazono, and C.H. Heldin. 1994. Identification and characterization of LTBP-2, a novel latent transforming growth factor-beta-binding protein. J Biol Chem. 269:32469-32478.
Moretti, F.A., A.K. Chauhan, A. Iaconcig, F. Porro, F.E. Baralle, and A.F. Muro. 2007. A major fraction of fibronectin present in the extracellular matrix of tissues is plasma-derived. J Biol Chem. 282:28057-28062.
Moustakas, A., S. Souchelnytskyi, and C.H. Heldin. 2001. Smad regulation in TGF-beta signal transduction. J Cell Sci. 114:4359-4369.
Munger, J.S., X. Huang, H. Kawakatsu, M.J. Griffiths, S.L. Dalton, J. Wu, J.F. Pittet, N. Kaminski, C. Garat, M.A. Matthay, D.B. Rifkin, and D. Sheppard. 1999. The integrin alpha v beta 6 binds and activates latent TGF beta 1: a mechanism for regulating pulmonary inflammation and fibrosis. Cell. 96:319-328.
Muro, A.F., A.K. Chauhan, S. Gajovic, A. Iaconcig, F. Porro, G. Stanta, and F.E. Baralle. 2003. Regulated splicing of the fibronectin EDA exon is essential for proper skin wound healing and normal lifespan. J Cell Biol. 162:149-160.
Muro, A.F., F.A. Moretti, B.B. Moore, M. Yan, R.G. Atrasz, C.A. Wilke, K.R. Flaherty, F.J. Martinez, J.L. Tsui, D. Sheppard, F.E. Baralle, G.B. Toews, and E.S. White. 2008. An essential role for fibronectin extra type III domain A in pulmonary fibrosis. Am J Respir Crit Care Med. 177:638-645.
69
Neilson, E.G., D. Plieth, and C. Venkov. 2003. Epithelial-mesenchymal transitions and the intersecting cell fate of fibroblasts and metastatic cancer cells. Trans Am Clin Climatol Assoc. 114:87-100; discussion 100-101.
Nistala, H., S. Lee-Arteaga, S. Smaldone, G. Siciliano, L. Carta, R.N. Ono, G. Sengle, E. Arteaga-Solis, R. Levasseur, P. Ducy, L.Y. Sakai, G. Karsenty, and F. Ramirez. 2010. Fibrillin-1 and -2 differentially modulate endogenous TGF-beta and BMP bioavailability during bone formation. J Cell Biol. 190:1107-1121.
Okamura, Y., M. Watari, E.S. Jerud, D.W. Young, S.T. Ishizaka, J. Rose, J.C. Chow, and J.F. Strauss, 3rd. 2001. The extra domain A of fibronectin activates Toll-like receptor 4. J Biol Chem. 276:10229-10233.
Ornitz, D.M., and N. Itoh. 2001. Fibroblast growth factors. Genome Biol. 2:REVIEWS3005.
Paine, R., 3rd, and P.A. Ward. 1999. Cell adhesion molecules and pulmonary fibrosis. Am J Med. 107:268-279.
Palmer, E.L., C. Ruegg, R. Ferrando, R. Pytela, and D. Sheppard. 1993. Sequence and tissue distribution of the integrin alpha 9 subunit, a novel partner of beta 1 that is widely distributed in epithelia and muscle. J Cell Biol. 123:1289-1297.
Paszek, M.J., N. Zahir, K.R. Johnson, J.N. Lakins, G.I. Rozenberg, A. Gefen, C.A. Reinhart-King, S.S. Margulies, M. Dembo, D. Boettiger, D.A. Hammer, and V.M. Weaver. 2005. Tensional homeostasis and the malignant phenotype. Cancer Cell. 8:241-254.
Peters, D.M., L.M. Portz, J. Fullenwider, and D.F. Mosher. 1990. Co-assembly of plasma and cellular fibronectins into fibrils in human fibroblast cultures. J Cell Biol. 111:249-256.
Peters, T., A. Sindrilaru, B. Hinz, R. Hinrichs, A. Menke, E.A. Al-Azzeh, K. Holzwarth, T. Oreshkova, H. Wang, D. Kess, B. Walzog, S. Sulyok, C. Sunderkotter, W. Friedrich, M. Wlaschek, T. Krieg, and K. Scharffetter-Kochanek. 2005. Wound-healing defect of CD18(-/-) mice due to a decrease in TGF-beta1 and myofibroblast differentiation. EMBO J. 24:3400-3410.
Quaggin, S.E., and A. Kapus. 2011. Scar wars: mapping the fate of epithelial-mesenchymal-myofibroblast transition. Kidney Int. 80:41-50.
Radnoff, O.D., and H. Saito. 1975. Inhibition of Hageman factor, plasma thromboplastin antecedent, thrombin and other clotting factors by phenylglyoxal hydrate (38500). Proc Soc Exp Biol Med. 148:177-182.
Ramirez, F., and D.B. Rifkin. 2009. Extracellular microfibrils: contextual platforms for TGFbeta and BMP signaling. Curr Opin Cell Biol. 21:616-622.
Ramirez, F., and L.Y. Sakai. 2010. Biogenesis and function of fibrillin assemblies. Cell Tissue Res. 339:71-82.
Rønnov-Jessen, L., and O.W. Petersen. 1993. Induction of alpha-smooth muscle actin by transforming growth factor-beta 1 in quiescent human breast gland fibroblasts. Implications for myofibroblast generation in breast neoplasia. Lab Invest. 68:696-707.
Ross, R. 1968. The fibroblast and wound repair. Biol Rev Camb Philos Soc. 43:51-96. Ruiz-Ortega, M., J. Rodriguez-Vita, E. Sanchez-Lopez, G. Carvajal, and J. Egido. 2007.
TGF-beta signaling in vascular fibrosis. Cardiovasc Res. 74:196-206.
70
Saharinen, J., J. Taipale, O. Monni, and J. Keski-Oja. 1998. Identification and characterization of a new latent transforming growth factor-beta-binding protein, LTBP-4. J Biol Chem. 273:18459-18469.
Satoi, S., Y. Hiramatsu, H. Kitade, A.H. Kwon, K. Matsui, K. Miyashita, E. Sakashita, K. Sekiguchi, H. Takahashi, and Y. Kamiyama. 1999. Different responses to surgical stress between extra domain A+ and plasma fibronectins. Clin Exp Pharmacol Physiol. 26:225-229.
Schaffer, M.R., P.A. Efron, F.J. Thornton, K. Klingel, S.S. Gross, and A. Barbul. 1997. Nitric oxide, an autocrine regulator of wound fibroblast synthetic function. J Immunol. 158:2375-2381.
Schubert, C., E. Christophers, O. Swensson, and T. Isei. 1989. Transendothelial cell diapedesis of neutrophils in inflamed human skin. Arch Dermatol Res. 281:475-481.
Schwarzbauer, J.E., J.W. Tamkun, I.R. Lemischka, and R.O. Hynes. 1983. Three different fibronectin mRNAs arise by alternative splicing within the coding region. Cell. 35:421-431.
Serhan, C.N., and J. Savill. 2005. Resolution of inflammation: the beginning programs the end. Nat Immunol. 6:1191-1197.
Serini, G., M.L. Bochaton-Piallat, P. Ropraz, A. Geinoz, L. Borsi, L. Zardi, and G. Gabbiani. 1998b. The fibronectin domain ED-A is crucial for myofibroblastic phenotype induction by transforming growth factor-beta1. J Cell Biol. 142:873-881.
Serini, G., and G. Gabbiani. 1996. Modulation of alpha-smooth muscle actin expression in fibroblasts by transforming growth factor-beta isoforms: an in vivo and in vitro study. Wound Rep Reg. 4:278-287.
Serini, G., and G. Gabbiani. 1999a. Mechanisms of myofibroblast activity and phenotypic modulation. Exp Cell Res. 250:273-283.
Shi, M., J. Zhu, R. Wang, X. Chen, L. Mi, T. Walz, and T.A. Springer. 2011. Latent TGF-beta structure and activation. Nature. 474:343-349.
Shinde, A.V., C. Bystroff, C. Wang, M.G. Vogelezang, P.A. Vincent, R.O. Hynes, and L. Van De Water. 2008. Identification of the peptide sequences within the EIIIA (EDA) segment of fibronectin that mediate integrin alpha9beta1-dependent cellular activities. J Biol Chem. 283:2858-2870.
Shipley, J.M., R.P. Mecham, E. Maus, J. Bonadio, J. Rosenbloom, R.T. McCarthy, M.L. Baumann, C. Frankfater, F. Segade, and S.D. Shapiro. 2000. Developmental expression of latent transforming growth factor beta binding protein 2 and its requirement early in mouse development. Mol Cell Biol. 20:4879-4887.
Singer, A.J., and R.A. Clark. 1999. Cutaneous wound healing. N Engl J Med. 341:738-746.
Strieter, R.M., E.C. Keeley, M.D. Burdick, and B. Mehrad. 2009. The role of circulating mesenchymal progenitor cells, fibrocytes, in promoting pulmonary fibrosis. Trans Am Clin Climatol Assoc. 120:49-59.
Szpaderska, A.M., J.D. Zuckerman, and L.A. DiPietro. 2003. Differential injury responses in oral mucosal and cutaneous wounds. J Dent Res. 82:621-626.
Thannickal, V.J., G.B. Toews, E.S. White, J.P. Lynch, 3rd, and F.J. Martinez. 2004. Mechanisms of pulmonary fibrosis. Annu Rev Med. 55:395-417.
71
Theoharides, T.C., K.D. Alysandratos, A. Angelidou, D.A. Delivanis, N. Sismanopoulos, B. Zhang, S. Asadi, M. Vasiadi, Z. Weng, A. Miniati, and D. Kalogeromitros. 2012. Mast cells and inflammation. Biochim Biophys Acta. 1822:21-33.
Thiery, J.P., H. Acloque, R.Y. Huang, and M.A. Nieto. 2009. Epithelial-mesenchymal transitions in development and disease. Cell. 139:871-890.
Todorovic, V., V. Jurukovski, Y. Chen, L. Fontana, B. Dabovic, and D.B. Rifkin. 2005. Latent TGF-beta binding proteins. Int J Biochem Cell Biol. 37:38-41.
Tomasek, J.J., G. Gabbiani, B. Hinz, C. Chaponnier, and R.A. Brown. 2002. Myofibroblasts and mechano-regulation of connective tissue remodelling. Nat Rev Mol Cell Biol. 3:349-363.
Unsold, C., M. Hyytiainen, L. Bruckner-Tuderman, and J. Keski-Oja. 2001. Latent TGF-beta binding protein LTBP-1 contains three potential extracellular matrix interacting domains. J Cell Sci. 114:187-197.
van de Loo, F.A., and W.B. van den Berg. 2009. Immunocytokines: the long awaited therapeutic magic bullet in rheumatoid arthritis? Arthritis Res Ther. 11:132.
van der Rhee, H.J., C.P. van der Burgh-de Winter, and W.T. Daems. 1979. The differentiation of monocytes into macrophages, epithelioid cells, and multinucleated giant cells in subcutaneous granulomas. II. Peroxidatic activity. Cell Tissue Res. 197:379-396.
Verhamme, P., and M.F. Hoylaerts. 2009. Hemostasis and inflammation: two of a kind? Thromb J. 7:15.
Wang, J.M., A. Sica, G. Peri, S. Walter, I.M. Padura, P. Libby, M. Ceska, I. Lindley, F. Colotta, and A. Mantovani. 1991. Expression of monocyte chemotactic protein and interleukin-8 by cytokine-activated human vascular smooth muscle cells. Arterioscler Thromb. 11:1166-1174.
Wardlaw, A.J., R. Moqbel, O. Cromwell, and A.B. Kay. 1986. Platelet-activating factor. A potent chemotactic and chemokinetic factor for human eosinophils. J Clin Invest. 78:1701-1706.
Werner, S., and R. Grose. 2003. Regulation of wound healing by growth factors and cytokines. Physiol Rev. 83:835-870.
White, E.S. 2006. Infliximab in sarcoidosis: more answers or more questions? Am J Respir Crit Care Med. 174:732-733.
White, E.S., R.G. Atrasz, B. Hu, S.H. Phan, V. Stambolic, T.W. Mak, C.M. Hogaboam, K.R. Flaherty, F.J. Martinez, C.D. Kontos, and G.B. Toews. 2006. Negative regulation of myofibroblast differentiation by PTEN (Phosphatase and Tensin Homolog Deleted on chromosome 10). Am J Respir Crit Care Med. 173:112-121.
White, E.S., F.E. Baralle, and A.F. Muro. 2008. New insights into form and function of fibronectin splice variants. J Pathol. 216:1-14.
White, E.S., M.H. Lazar, and V.J. Thannickal. 2003. Pathogenetic mechanisms in usual interstitial pneumonia/idiopathic pulmonary fibrosis. J Pathol. 201:343-354.
White, E.S., R.L. Sagana, A.J. Booth, M. Yan, A.M. Cornett, C.A. Bloomheart, J.L. Tsui, C.A. Wilke, B.B. Moore, J.D. Ritzenthaler, J. Roman, and A.F. Muro. 2010. Control of fibroblast fibronectin expression and alternative splicing via the PI3K/Akt/mTOR pathway. Exp Cell Res. 316:2644-2653.
72
Williams, E.C., P.A. Janmey, R.B. Johnson, and D.F. Mosher. 1983. Fibronectin. Effect of disulfide bond reduction on its physical and functional properties. J Biol Chem. 258:5911-5914.
Wipff, P.J., and B. Hinz. 2008. Integrins and the activation of latent transforming growth factor beta1 - an intimate relationship. Eur J Cell Biol. 87:601-615.
Wipff, P.J., D.B. Rifkin, J.J. Meister, and B. Hinz. 2007. Myofibroblast contraction activates latent TGF-beta1 from the extracellular matrix. J Cell Biol. 179:1311-1323.
Wright, H.L., R.J. Moots, R.C. Bucknall, and S.W. Edwards. 2010. Neutrophil function in inflammation and inflammatory diseases. Rheumatology (Oxford). 49:1618-1631.
Wu, C., J.S. Bauer, R.L. Juliano, and J.A. McDonald. 1993. The alpha 5 beta 1 integrin fibronectin receptor, but not the alpha 5 cytoplasmic domain, functions in an early and essential step in fibronectin matrix assembly. J Biol Chem. 268:21883-21888.
Wynn, T.A. 2007. Common and unique mechanisms regulate fibrosis in various fibroproliferative diseases. J Clin Invest. 117:524-529.
Xia, P., and L.A. Culp. 1995. Adhesion activity in fibronectin's alternatively spliced domain EDa (EIIIA): complementarity to plasma fibronectin functions. Exp Cell Res. 217:517-527.
Xu, X.H., P.K. Shah, E. Faure, O. Equils, L. Thomas, M.C. Fishbein, D. Luthringer, X.P. Xu, T.B. Rajavashisth, J. Yano, S. Kaul, and M. Arditi. 2001. Toll-like receptor-4 is expressed by macrophages in murine and human lipid-rich atherosclerotic plaques and upregulated by oxidized LDL. Circulation. 104:3103-3108.
Yin, W., E. Smiley, J. Germiller, R.P. Mecham, J.B. Florer, R.J. Wenstrup, and J. Bonadio. 1995. Isolation of a novel latent transforming growth factor-beta binding protein gene (LTBP-3). J Biol Chem. 270:10147-10160.
Zeisberg, M., and J.S. Duffield. 2010. Resolved: EMT produces fibroblasts in the kidney. J Am Soc Nephrol. 21:1247-1253.
Zhou, Y., J.S. Hagood, B. Lu, W.D. Merryman, and J.E. Murphy-Ullrich. 2010. Thy-1-integrin alphavbeta5 interactions inhibit lung fibroblast contraction-induced latent TGF-beta1 activation and myofibroblast differentiation. J Biol Chem.