Perlecan, an extracellular matrix protein, mediates vascular smooth muscle cell quiescence

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Perlecan, an extracellular matrix protein, mediates vascular smooth muscle cell quiescence
Walker, Heather A
Place of Publication:
Denver, CO
University of Colorado Denver
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60 leaves : illustrations ; 28 cm


Subjects / Keywords:
Extracellular matrix proteins ( lcsh )
Vascular smooth muscle ( lcsh )
Muscle cells ( lcsh )
Extracellular matrix proteins ( fast )
Muscle cells ( fast )
Vascular smooth muscle ( fast )
bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )


Includes bibliographical references (leaves 57-60).
General Note:
Department of Integrative Biology
Statement of Responsibility:
by Heather A. Walker.

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|University of Colorado Denver
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|Auraria Library
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47826269 ( OCLC )
LD1190.L45 2001m .W34 ( lcc )

Full Text
Heather A. Walker
B.A., University of Colorado, Boulder, 1995
A thesis submitted to the
University of Colorado at Denver
in partial fulfillment
of the requirements for the degree of
Master of Arts

The thesis for the Master of Arts
degree by
Heather A. Walker


Heather A. Walker (MA, Biology)
Perlecan, an Extracellular Matrix Protein, Mediates Vascular Smooth Muscle Cell
Thesis directed by Assistant Professor Mary CM Weiser-Evans
During embryonic development and after vascular injury, vascular smooth muscle
cells (SMC) exhibit high rates of proliferation, which is a major contributor to blood
vessel maturation and vascular remodeling. However, in a mature, uninjured vessel,
SMC are highly quiescent. Components of the SMC basement membrane, in
particular, perlecan heparan sulfate, are believed to contribute to vascular SMC
growth suppression by inhibiting the activation of growth-promoting signaling
pathways. Understanding this suppression is important for reversing the mechanisms
involved in the pathogenesis of a variety of vascular diseases. The mechanism
inducing this growth suppressive response and signaling events contributing to this
inhibition are poorly understood. For the studies described here, adult rat SMC
isolated from uninjured aortas were plated on individual matrices of perlecan, as well
as stimulatory matrices such as fibronectin, laminin, collagen I and plastic. Using
various techniques we examined the effects of various matrix proteins on SMC
growth control. The levels and activities of several growth-associated signaling
proteins, such as FAK and ERK 1/2, were also analyzed. Activation of FAK and ERK
1/2 was found to be decreased when cells were plated on perlecan or on intact
basement membranes. In contrast, FAK and ERK 1/2 were significantly activated
upon plating SMC on the above-mentioned growth-stimulatory matrix proteins.
Similar to the culture experiments, in vivo studies demonstrated that SMC in intact,
uninjured vessels, surrounded by perlecan-enriched basement membranes, have little
activated FAK. We next studied the effect of perlecan-enriched basement membranes
on Ras-GTP-transfected SMC, cells that express constitutively active MAP Kinase

signaling. Compared to empty vector-transfected SMC, we found that mitogen-
stimulated replication was not suppressed by intact basement membranes. Therefore,
the growth inhibitory effect of perlecan is initiated upstream of Ras. Finally, using a
neutralizing antibody to perlecan, we found that, in addition to the growth-inhibitory
effects of exogenous perlecan, endogenously-produced perlecan significantly
attenuates mitogen-induced growth of SMC. Overall, Perlecan was found to actively
suppress SMC growth by inhibiting integrin-mediated, MAP Kinase-dependent
This abstract accurately represents the content of the candidates thesis. I recommend
its publication,
Mary CM Weiser-Evans

I dedicate this thesis to my husband for his steadfast support and understanding
while I was pursuing this degree.

Many thanks to my advisor, Mary Weiser-Evans for her continued patience and
support during these past two years. I also wish to thank the other members of our
laboratory for advice and assistance in pursuing this degree. Lastly, I would like to
thank all the members of my committee for their aid and understanding.

1. INTRODUCTION..............................................1
2. BACKGROUND/REVIEW OF LITERATURE...........................8
3. MATERIALS AND METHODS....................................23
Cell lysate preparation...............................23
Immunostain protocol..................................25
Western Blotting and transfers........................30
MAP Kinase assay......................................32
4. RESULTS/DATA.............................................34
Plating cells on perlecan inhibits MAP K activation...34
Plating cells on perlecan inhibits FAK activation.....38
Perlecans inhibitory effect is overridden by MAP Kinase
FAK activation is low in an uninjured in vivo system .44
Plating cells on perlecan decreases focal contacts....45
Plating cells on perlecan inhibits GF-R activation....48

Abolishing endogenous perlecan decreases SMC growth 49
5. CONCLUSION............................51

1.1a Cross-section of vessel (electron micrograph).......................1
1.1b Schematic cross-section of arterial wall............................1
1.2 Electron micrograph of diseased vessel...............................3
1.3 Role of perlecan; mature cell vs. replicating cell...................5
2.1 Schematic of cellular basement membrane...............................9
2.2 Schematic of pelecan molecule.......................................10
2.3 Schematic of cell growth signaling pathway (two hit)................12
2.4 Schematic of cell inhibitory signaling pathway......................13
2.5 Integrin structure..................................................14
2.6 Focal Adhesion Kinase structure.....................................19
4.1 Immunostain-P-ERK and DAPI after 30 min..............................35
4.2 Immunostain-FN after 24 h, multiple stains..........................36
4.3 Immunostain-Perlecan after 24 h, multiple stains....................37
4.4 Western Blot on Matrigel and plastic and IP with P-FAK..............38
4.5 Western Blot on PI, FN, PN, total FAK and P-FAK.....................40
4.6 IP with FAK, probed with P-FAK......................................40
4.7 Schematic of Ras-GTP pathway........................................41
4.8 Bar graphs-Map Kinase activation and Cell number, Ras-GTP cells.....42
4.9 Western of Ras-GTP cells, probed with P-FAK and total FAK...........44
4.10 Western blot, in vivo and susp, P-FAK and total FAK.................45
4.11 Immunostain-Phalloidin and DAPI on Perl and FN......................46
4.12 Immunostain-P-FAK, Paxillin and DAPI on Perl and FN................47
4.13 Western Blot with and without PDGF-BB on FN, Mat and Susp..........49
4.14 Bar graph with total cell number after Day 0, 3, 5 with and without anti-PN 50

3.1 Immunostain antibodies with dilutions................................26
3.2 PAGE gel recipes.....................................................28
3.3 PAGE and Western Blotting buffer recipes.............................28
3.4 Western Blotting primary antibodies and sources......................30
3.5 Recipes for MAP Kinase Assay Lysis Buffer and Mix....................33

Smooth Muscle Cell (SMC) replication and extracellular matrix (EMC) pro-
tein expression both have fundamental roles in the normal development of the vas-
culature as well as in the process of restenosis (Belknap et al., 1999, Pauly et al.,
1992, Schwartz et al., 1995). The anatomy of a blood vessel can be seen in Figure
1.1a. In a normal adult aorta, the vessel has distinct layers with a small intimal diam-
endothelial lining smooth muscle collagen
loose connective tissue
smooth muscle
elastic lamina (elastin fibers)
endothelial lining
basal lamina
Figure 1A)
Schematic drawing cross-sectional view of a normal adult vessel.
Figure 1B)
Scanning electron micrograph of a cross-section through an artery show-
ing the inner layer of endothelial cells, the smooth muscle layer, and the
collagenous connective tissue (Alberts, etal., 1994).

eter, which lies on top of the endothelial cell layer (Figure 1.1b). In a normal adult
vessel, the SMC are highly differentiated and act in a contractile manner. These
SMC are extremely quiescent exhibiting replication rates as low as < 0.05% per day
and they are resistant to growth stimulation (Cook et al., 1993, Pauly et al., 1992,
Clowes AW, et al, 1983). However, in a diseased or injured vessel, there is a great
increase in the thickening of the intimal layer, which produces a decreased lumen
area for blood flow (See Figure 1.2). As a result of an injury to an adult vessel,
some of these SMC begin to replicate and usually migrate into the lumen of the ves-
sel. This process is called restenosis, which is simply the re-growth of the vessel
as the new intima or neointima surrounds the medial SMC in the vessel lumen.
The rate restenosis can be as high as 20-60% of all patients undergoing transluminal
angioplasties or other forms of mechanical intervention per treatment of atheroscle-
rosis. The mechanism of restenosis itself, as well as any methods to prevent it,
remain unclear (Guarda, E et al, 1996). It has been shown that neointimal SMCs,
collected 15 days after lesion, proliferate more actively than do medial cells in the
presence of the same amounts of serum, and moreover, some of these SMCs are
capable of growing in the absence of serum growth factors (Orlandi, A, et al., 1993)
This intimal thickening and decreased diameter of the lumen can compromise the
blood flow area and can be very dangerous for reasons of increased blood pressure,
onset of angina, vasospasms and even increased diabeters mellitus complications
(Lascalzo J, 1992).
As one example, the process of atherosclerosis is thought to be initiated by
injury to the arterial wall, followed by a reparative response by the vascular SMC.
More specifically, this response consists of proliferation and migration of SMC with-

Plate V. Luminal obstruction by excessive intimal hyperplasia (elastin-van
Gieson; 48.5 x).
Figure 1.2)
Electron micrograph of abnormal diseased vessel with fatty
deposits and decreased area of the lumen where blood flows.

in the intima of the artery, causing great intimal expansion as seen in atherosclerotic
plaques (Ohno T, et al, 1994, Schwartz, et al., 1995).
There are numerous causes for arterial neointimal formation with the most
common being the disease states of atherosclerosis, hypertention, infection (often
viral), or mechanical injuries such as transluminal angioplasties, stent placement, or
vascular grafts as mentioned above. Other injuries shown to result in neointimal for-
mation include 1) radiation 2) electrical stimulation 3) wrapping the vessel (Ross et
al., 1993, Schwartz et al., 1995). After an insult to the vessel the SMCs function
differently. The cells are now de-differentiated, begin to migrate to the intima, show
increased replication, and secrete Extracellular Matrix (ECM) proteins such as colla-
gen I and fibronectin. These changes all are believed to contribute to the formation
of a neointima.
While the mature SMCs of the vasculature are known to be quiescent and
resistant to growth stimulation, the factors which maintain this quiescence in the
normal arterial wall are unknown. The goal of this project is to understand the
mechanism(s) underlying the establishment and maintenance of SMC quiescence in
the mature, uninjured vessel wall. Our studies centered on the role of perlecan, a
protein found in high abundance in the SMC basement membrane that directly sur-
rounds the cell. We propose a hypothetical role for this protein for the control of
SMC growth.
A fully mature SMC, as previously mentioned, is replication resistant, very
quiescent in vivo, and its growth is serum-dependent in vitro. These cells also have
an intact matrix that surrounds each cell called the pericellular basement membrane
(Heickendorff, 1989). This cellular matrix includes many ECM proteins, such as

fibronectin, collagen I, collagen IV, perlecan, entactin and laminin. Collagen IV,
fibronectin and perlecan are the most abundant SMC basement membrane proteins
(Heickendorff, 1989, Weiser et al, 1997). This is the profile of an adult SMC after
development has ceased. On the other hand, upon an injury to the matrix, such as
physical, proteolytic, or disease-driven injury, the cells begin to replicate at high lev-
els, and exhibit autonomous replication in vitro (defined as the ability to replicate in
the absence of exogenous growth factors or serum) (See figure 1.3).
How is this occurring? If these cells are replicating at high rates, the accu-
mulations of intimal smooth muscle occurs, which is known to cause numerous
problems leading to atherosclerotic lesions. In addition to cell growth, this phenotyp-
ic modulation leads also to an increased production of various extracellular matrix
Hypothetical role for SMC basement membrane perlecan
in the control of SMC growth
Fully Mature SMC
Replicating SMC
Matrix Injury
(physical, proteolytic,
Replication Resistant
Replication Competent
Very quiescent in vivo
Serum-dependent growth in vitro
Active repression of FAK-
mediated MAPK activation
High in vivo replication rates
Autonomous replication in vivo
Activated FAK-induced MAPK
Figure 1.3)
Schematic illustrating the two states of a cell with an intact
basement membrane and a disrupted bsement membrane.

molecules (Ohno T, et al., 1994). While we do not know whether atherosclerosis is
the cause or the effect of this cell accumulation, we do know that replicating as well
as quiescent phenotypes of the cells must be better understood in order to maintain a
healthy vessel. Previous data from our laboratory have provided evidence that per-
lecan, the predominant heparan sulfate proteoglycan in the basement membrane, has
a role in producing an inhibitory response of the adult SMC.
In addition to increased cell growth that occurs in an injured vessel comes
the increased production of Extracellular Matrix molecules (ECM) by SMCs. In an
effort to better understand the mechanisms duing cell replication and ECM produc-
tion, it has been shown that antiproliferative therapies can be effective in preventing
the development of intimal lesions in the smooth muscle cells (Assoian and
Marcantonio, 1996, Ohno et al., 1994, Chang et al., 1995).
In one study of SMC and injury, the vessels were injured and levels of
mRNA transcripts for ECM proteins were measured. After one week of injury, tran-
scripts for some heparan sulfate proteoglycans (HSPG), one chondroitin sulfate pro-
teoglycan and one dermatan sulfate proteoglycan were all greatly increased. This
illustrates that ECM deposition does occur and is a late event in response to injury
(Nikkari et al., 1994).
Others have shown that ECM components regulate development and differ-
entiation of cells including SMC, and have great effects on migration and growth as
well (Adams and Watt, 1993, Hedin et al., 1988). There are three mechanisms by
which extracellular matrix can regulate cell behavior:
1) through changes in the composition of ECM 2) through synergistic interactions

between growth factors (GFs) and matrix molecules 3) through the cell surface
receptors that mediate the ECM components. ECM does not simply consist of the
individual molecules secreted by and surrounding cells, but is a complex arrange-
ment that is dictated by the composite organization of the supramolecular structure.
Therefore, the questions that arise are: what ECM molecules control or contribute to
this neointimal formation? What can this formation after injury tell us about the
critical mechanisms underlying arterial pathology in aging atherosclerosis or
restenosis and in their prevention (Schwartz, et al., 1995)?
In other words, what are the underlying molecular mechanisms that abate this
neointimal layer formation and therefore diminish restenosis and diseased vessels?
We believe a significant contributor to the inhibition of neointima formation and to
the quiescence of SMC in an uninjured adult vessel, is the heparan sulfate proteogly-
can, perlecan.

The Extracellular Matrix
The molecules of the ECM exist around and are secreted by most cells of
multicellular organisms. The ECM acts as a structural support providing anchorage
of the SMC to its environment, as well as fundamental signals regulating cellular
growth, metabolism, and differentiation (Adams and Watt, 1993, Yamada, 1998).
The ECM is also involved in cell migration and motility as well as maintaining elas-
tic properties of the vessel wall (Heidendorff, 1989). In recent discovery, the bind-
ing of specific ECM molecules to their plasma membrane receptors activates signal-
cascading responses that can include activation of various tyrosine and serine-threo-
nine kinase families, MAP Kinase systems, and ion fluxes. The recent purification
of the individual ECM molecules has allowed the studies to flourish along with the
realization that ECM components may be responsible for many crucial cell-biologi-
cal functions. (Yamada, 1998).
The ECM macromolecules are involved in supramolecular assemblies where
their biological properties are modified by the molecules with which they interact. It
is very difficult to determine the functional properties of an elaborate matrix without
studying the individual components. However, the functional complexity of the

basement membrane as a whole is much greater than the sum of its individual parts.
The ECM is also very functionally diverse. Many molecules are designed to be
rigid, others elastic, some sticky, others wet. The individual molecules are highly
specialized and also have modular designs that are involved in diverse roles
(Yamada, 1998).
Perlecan is a member of the Proteoglycan protein family with approximately
2-15 heparan sulfate glucosaminoglycan chains (See Figure 2.1). It has a molecular
weight of about 600,000 daltons and is located mainly in the basal laminae sur-
rounding each individual cell. It has been shown to function as a structural
Figure 2.1)
Representation of a cellular basement membrane. Perlecan is
shown in green (Alberts etal., 1994)

Basement Membrane (BM) component along with fibronectin, laminin, and entactin,
(Figure 2.1) (Alberts et al., 1994). Figure 2.1 illustrates a cellular basement mem-
brane. Perlecan, along with the other proteins, is involved in a network of scaffold-
ing that clearly provides structure for the cell. The perlecan molecule itself has five
domains and has been implicated in the control of SMC growth and differentiation
(Weiser et al., 1996, Ottlinger et al, 1993, Lankes, et al., 1988) (See Figure 2.2).
The proteoglycans (PGs) are ubiquitous macromolecules that contain gly-
cosaminoglycan (GAG) side chains attached to a core polypeptide chain. PGs are
found mainly outside of the cell as a part of the ECM and are prevelent in most con-
Five domains:
Domain I probable heparan sulfate attachment site
-Domain II homologous to the LDL-binding domain of the LDL receptor
-Domain III and V homologies to Laminin
Domain IV Numerous Ig-like repeats
---------- II
Domain I
Figure 2.2)
Schematic drawing of the perlecan molecule (human) depicting its five
domains (lozzo et al., 1994).

nective tissue. The GAGs are made up of repeating disaccharides and are high in
molecular weight, highly sulfated and negatively charged. The GAG and PG accu-
mulation is responsible for overall properties of tissues such as hydration, elasticity,
and permeability. The GAGs and PGs associate with other proteins in the ECM,
such as collagen and fibronectin as well as with protein meshworks or a number of
proteins. These factors enable the PGs to be linked with adhesion and motility as
well. (Alberts et al., 1994, Forsten, KE et al., 1997, Ottlinger ME, et al., 1993,
Fritze LM, et al., 1985).
Heparin is a synthetic mucopolysaccharide that closely resembles the
heparan sulfate side chains of proteoglycans. Therefore, it has been used widely in
HS studies. It has been demonstrated to work as an anticoagulant (Alberts., et al.,
1994). Also, Heparin has been found to be a potent antiproliferative molecule for
vascular smooth muscle cells both in vivo and in vitro, typically blocking the prolif-
eration phase of the cell cycle (Castellot et al., 1982, Lankes et al., 1988). Expression
of growth related genes, such as c-fos, c-myc, and c-myb are inhibited by heparin.
Heparin has also been found to inhibit the activation of the MAP Kinases in
response to mitogen stimulation and following arterial injury (Clowes AW and
Kamovsky MJ, 1977, Ottlinger ME et al., 1993). Clowes and Kamovsky (1977)
showed that exogenous heparin could inhibit SMC proliferation in response to
removal of the endothelial layer in a vessel. Fritze et al. (1985) demonstrated that
SMC are capable of controlling their own growth by the synthesis of a specific
form of heparan sulfate with an antiproliferative potency. Campbell et al. (1992)
suggested that removal of the HS from the surface of SMC (as well as its degrada-
tion by macrophage protease secretion) temporarily interrupts the normal quiescent

state of SMC and induces a smooth muscle phenotypic change. Because of per-
lecans large HS components, it has been targeted as a potential inhibitor of SMC
In order for cell growth to occur, it has been theorized that a cell needs two
hits. Normal adult cell growth requires stimulation by both growth factors and
adhesion to its ECM (Howe et al., 1998, Oktay et al., 1999, Renshaw et al., 1997,
1999; see figure 2.3). Through the synergism of these pathways, the cell will
actively grow (discussed in more detail below). We believe that perlecan, a basement
membrane protein, inhibits SMC growth by actively inhibiting the integrin-specific
FN, Col IV
LN, Col I
Cell Growth
The Two-Hit Activation
Normal cell growth requires
stimulation by both growth
factors and adhesion to the
Howe et al., 1998,
Renshaw et al., 1997,1999
Oktay et at., 1999
Figure 2.3)
Aschmatic illustrating the pathway for Cell Growth. There is a "two hit"
activation theory stating that normal cell growth requires stimulation by
both growth factors and adhesion to the ECM. GF=growth factor, GF-R=
growth factor receptor, FAK= Focal Adhesion Kinase, ERK 1/2= the MAP
Kinases, PI3K= Phosphoinositol 3, Kinase, PKC= Protein Kinase C.

Growth Inhibition
The Integrin pathway is
suppressed, and the SMC are
now rendered Growth Factor
Howe et al, 1998,
Renshaw et al, 1997, 1999
Oktay et aL, 1999
Figure 2.4)
Schmatic showing the potential suppressive points in the perlecan mediat-
ed Growth Inhibition pathway. Perlecan might inhibit SMC growth 1) at the
integrin level on the extracellular side of the cell 2) by downregulating pro-
teins upstream of the MAP Kinase proteins or 3) directly, by down regulating
MAP Kinase proteins. The integrin pathway is suppresed by perlecan, and
the SMC are now rendered growth factor unresponsive.

signaling pathways. In this model, we propose that active suppression of the inte-
grin signaling pathway by perlecan renders SMC growth factor unresponsive and
therefore, replication resistant (See figure 2.4).
The integrins are heterodimeric transmembrane linker proteins that are the
main receptors that bind to the extracellular matrix (Figure 2.5). The proteins such
as fibronectin, laminin, perlecan and collagen bind to the integrin receptors with low
affinity, but many integrins are bound at once, so that the cells still maintain a fluidi-
ty in their environments. Some integrins bind to many matrix proteins, whereas oth-
matrix binding
Figure 2.5)
The subunit structure of an integrin cell-
surface matrix receptor (Alberts, etal.,
talin an
a-actinin binding
10 nm

ers bind to only one. About twenty heterodimers have been defined made from all
types of a and P combinations (Alberts, et al., 1996).
The receptor consists of two subunits, a and p, which are associated via a
non-covalent bond, and which are both involved in binding matrix proteins. Either
the cytoplasmic tail of the a or P subunit binds to the cytoskeleton and also to the
signaling proteins that exist around the membrane. Hence, the integrins are the
mediators between the ECM and the cytoskeleton inside the cell. Most integrins
connect to actin filaments. After the integrin binds to its ligand outside the ceil, the
P chains cytoplasmic tail binds to talin or another protein commencing the focal
contacts that cluster at the membrane. Therefore, the integrins are now considered
to function not only as adhesive receptors of the extracellular proteins, but also act
as signaling receptors that are vital to vascular cells (Alberts, et al., 1996, Schwartz,
et al., 1995).
The integrins are necessary for cell growth, differentiation, and survival in
many cell types, including the SMC. More specifically, integrins have been shown
to play a role in development, angiogensis, cell migration in response to injury and
extracellular matrix assembly. These cell behaviors can most likely be attributed to
the activation of FAK, Src, IP3 and the MAP Kinases, all shown to be involved in
integrin-triggered phenomena cell signaling events (Shattil SJ and Ginsberg, 1997).
Cell Signaling
All animal cells contain elaborate systems of proteins that allow cells to
respond to signals from each other. This system includes cell-surface and intracellu-
lar receptor proteins, protein kinases, protein phosphatases, GTP-binding proteins

and many intracellular proteins with which these signaling proteins interact. These
signals could be derived from secreted molecules or from plasma-membrane bound
molecules. The extracellular signaling molecules bind to either cell-surface recep-
tors or to intracellular receptors. It is the combinations of signaling molecules that
enable an animal (or cell) to control the behavior of its cells in a highly specific way
using a limited diversity of such molecules: hundreds of such signals can be used in
millions of combinations. Different cells can respond differently to the same chemi-
cal signals (Alberts, et al., 1994). Many of the proteins involved in cell signaling
related to SMC growth control will be described below.
In general, mitogenesis is initiated when the membrane bound receptors
(such as integrins) are activated, they trigger phosphate group additions to a con-
glomeration of intracellular proteins and a phosphorylation cascade begins. The sig-
nals that are received at the surface of the cell are usually relayed to the nucleus,
where they alter the expression of specific genes and thus change the cells course of
action. This cascade of signaling molecules form elaborate relay systems. The
proteins are of two types: proteins that become phosphorylated by protein kinases
and proteins that are induced to bind GTP when the signal arrives. In either situa-
tion, the proteins lose the phosphates when the signal decays, or gain one or more
phosphates in the activated state. The phosphorylation cascade can now begin as
these activated proteins, in turn, cause the phosphorylation of proteins downstream
towards the nucleus (Refer to Figures 2.3 and 2.4 as examples).
The two main types of protein kinases involved in the phosphorylation cas-
cades are the serine/threonine kinases, which phosphorylate target proteins on serine
and threonine residues and the tyrosine kinases which phosphorylate target proteins

on tyrosine residues. Combinations of these signals, rather than a lone signal, often
stimulate the more complex cell behaviors, such as proliferation and survival. The
cell must integrate the information coming from the separate signals so as to make
the proper responseto live or die, or to proliferate or stay quiescent. This integra-
tion is often a mystery and seems to depend on the interactions between different
protein phosphorylation cascades, which are activated by various extracellular sig-
The Ras proteins are a crucial link in the intracellular signaling pathways
which are activated by the receptor tyrosine kinases. Ras in turn, activates a
serine/threonine phosphorylation cascade that then activates the MAP Kinases
(Mitogen-Activated Protein Kinases, also known as the Extracellular Signal
Regulated Kinases or ERKs). The MAP Kinases are one family of kinases that con-
tain at least five members and play a very fundamental role in cell signaling, often
integrating multiple signals from various second messengers. The MAP Kinase acti-
vation involves numerous phosphorylation steps of serine/threonine phosphate addi-
tions and are initially turned on by a wide variety of extracellular proliferation and
differentiation-inducing signals. Upon activation of MAP Kinases, which are rapidly
activated in response to growth factor stimulation, they relay signals downstream by
phosphorylating a wide range of proteins in the cell, including other protein kinases
and gene regulatory proteins (Velvarde, et al., 1999). Once activated, the MAP
Kinases migrate from the cytosol into the nucleus and finally activate c-fos gene
transcription, an immediate early gene involved in cell growth. In addition, MAP
Kinases may activate the Jun protein, which combines with the newly made Fos pro-
tein to form an active gene regulatory protein called AP-1. This protein then turns

on additional genes, although its exact role in stimulating cell proliferating remains
to be defined.
The MAP kinase pathway is a major regulator of both oncogenic and normal
growth. It has been well established that normal cell growth requires cell adhesion to
extracellular matrix proteins as well as stimulation by growth factors (Clark and
Brugge, 1995, Renshaw et al., 1997). Typically, the integrins are the main transduc-
ers of ECM-mediated intracellular signals. Many researchers now report that activa-
tion of MAP kinase pathways, and specifically ERK-1 and ERK-2, by growth fac-
tors is strongly dependent on cell adhesion to extracellular matrix proteins via inte-
grin receptors (Alpin AE and Juliano RL, 1999, Assoican RK and Marcantonio, EE,
1996, Howe et al, 1998, Renshaw et al., 1997).
Focal Adhesion Kinase
Focal Adhesion Kinase (FAK) is a protein tyrosine kinase which is associat-
ed with intracellular signaling cascades which are initiated when the integrin family
of cell adhesion molecules engage Extracellular Matrix molecules (ECM) (Ridyard
and Sanders, 1999).
The structure of the FAK protein is shown in Figure 2.6. FAK has six tyro-
sine residues which are identified as sites of tyrosine phosphorylation. The major
site of autophosphorylation is tyrosine residue 397 whereas other tyrosine sites are
often phosphorylated by Src as well as acting as the binding sites for the SH2 and
the SH3 (Src homology) domain of the Grb2 adaptor protein (Schaller et al., 1999).
The Focal Adhesion Targeting (FAT) domain contains tyrosine phosphorylation sites
and runs from the 925-950 residues and binds the SH2 and SH3 domains of paxillin
and Grb2. FRNK (FAK-related non-kinase), the C-terminal domain of FAK, is

397 407 576/577
W 1/
i i
D704; XD772
Figure 2.6)
Focal Adhesion Kinase Molecule. Y=Phosphotyrosine, S=Phosphoserine,
Blue=unique regions, Orange=4.1 Domain (35-368), Red=Kinase Domain
(422-680), Green=Proline-rich regions (712-733, 863-913), Yellow=Paxillin-
Binding Site (919-1042). (Schaefer, EM, Bressler-Hill, V, 1999)
expressed as a separate protein. It does not contain the tyrosine kinase region of
FAK and therefore, acts as an endogenous-competitive-inhibitor of FAK. The FAK
molecule is shown to have a plentitude of binding sites for other adaptor proteins;
this is believed to be one reason that FAK may have so many varying effects after its
FAK derives its name from the fact that it also localizes to the focal adhe-
sions or focal contacts in vitro where it interacts with potential substrates for
phosphorylation such as paxillin, which bind to other proteins to form intricate link-
ages found in this cell contact (Rodriguez-Femandez, 1999). At these focal adhe-
sions, FAK can interact with other proteins in this location to activate intracellular
events associated with cell adhesion, migration, differentiation and growth. FAK is

activated by a variety of ECM proteins that bind different integrins. Therefore, many
different matrices can promote activation of FAK (Renshaw et al., 1999). The in
vitro expression of FAK and its level of activation appear to be related to cell
spreading, cell differentiation, cell locomotion and cell death. These events can
occur in integrin or non-integrin mediated signaling through FAKs association with
Src or with the MAP Kinase pathway. All of these phenomena are critical during
development and FAK is expressed in embryonic cells, therefore, FAK is considered
to be essential for morphogenesis and the developmental process (Ridyard and
Sanders, 1999).
FAK is also found to be activated by other surface receptors besides the
adhesion receptors such as neuropeptides, catecholamines, receptor tyrosine kinases,
chemokines, and other cytokines, immunoglobulins, bioactive lipid neurotransmitters
and cannabinoid receptors. Other stimuli that induce phosphorylation of FAK
include the treatment of the cells with phorbol esters, neurotoxins, bioactive lipids
acting intracellularly, subjection of cells to fluid sheer stress, neuronal depolarization
and glucose stimulation (Rodriguez-Femandez, JL, 1999). Hence, multiple stimuli
acting on distinct cell surface receptors that trigger distinct cytoplasmic signaling
pathways, converge to commence a common response, the tyrosine phosphorylation
of FAK (Schaller, et al., 1999). Moreover, the integrin signal leading to the activa-
tion of MAP Kinase and subsequent cell growth has been shown to be mediated by
FAK. Further, it has been shown that FAK is recruited to sites on integrin and
growth factor receptor clustering. Therefore, it is likely that growth factor and inte-
grin signaling pathways converge at the level of FAK for a synergistic effect on

The tyrosine phosphorylation of FAK is also modulated by receptor cluster-
ing after integrins have been activated. FAK is considered to be a candidate mole-
cule to transmit the signals from integrin receptors to start receptor oligomerization
(clustering). Rodriguez-Fernandez (1999) suggests that integrin receptor clustering
may be a common mechanism by which distinct factors may modulate tyrosine
phosphorylation of FAK. Therefore, disturbing integrin receptor clustering should
reduce the increase in FAK phosphorylation that is induced by receptor engagement
or by growth factor stimulation. By disrupting pre-formed integrin clustering in
focal contacts (for example, with cytochalasin D) or preventing focal contact forma-
tion (keeping cells in suspension), these treatments may inhibit FAK phosphoryla-
tion (Rodriguez-Femandez, JL, 1999).
In addition to its effects on cell growth, activation of FAK has been demon-
strated to interact with the actin cytoskeletal organization associated with cell migra-
tion. FAK activates paxillin with the subsequent activation of actin-binding proteins,
which leads to actin reorganization. This can be shown with phalloidin staining via
immunofluorescence. In addition, FAK may interact with other cytoplasmic signal-
ing pathways such as the Protein Kinase C (PKC) pathway or another mitogen-acti-
vated pathway (Gahtan et al., 1999).
Focal Adhesion Kinase is a pivotal protein that upon activation, may initiate
numerous cell signaling pathways. In conjunction with the integrin receptors, focal
adhesion activation causes receptor clustering and a subsequent cell signaling cascse
is started, leading to cell growth. Perlecan is proposed to inbibit the proliferative
cell cycle, involving FAK and potentially the MAP Kinases.
The mechanisms involving the establishment and continuity of SMC quies-

cence in a normal arterial wall are unestablished. The SMC basement membrane
and its components may contribute to the control of this SMC growth. Primarily, the
protein perlecan, a heparan sulfate proteoglycan in the ECM, may inhibit SMC
growth at three potential sites. This investigation examines the cell signaling and its
individual components that are involved in this inhibition of growth.

Preparation of Cell Lysates (matrix and non-matrix studies)
Many of the experiments performed involved the lysing of adult rat aortic
Smooth Muscle Cells (SMC) for the study of the cellular protein after the cells were
plated on various matrices, on plastic, or on glass. After the surgical removal of the
cells, they were grown via explant procedures to confluence. In the matrix studies,
the cells were plated on fibronectin (FN, 20 ug/ml), laminin (LN, 20 ug/ml), colla-
gen type I (Col I, 5 ug/ml), perlecan (PN, 5 ug/ml), collagen type IV, (Col IV, 20
ug/ml) or Matrigel (Mat, 50ul/cm^) for 1, 6, or 24 hours prior to lysing. These
matrices (except perlecan) were purchased from the Collaborative Biomedical
Company. Perlecan was supplied by J. Whitelock, CSIRO, Australia. For the non-
matrix studies, the cells wereplated on plastic and lysed after reaching confluency.
For some experiments, SMC were growth arrested by incubation in 0.1%
Newborn Calf Serum (NCS) in Dulbeccos Modified Eagle's Media (DMEM) or by
incubation in serum-free DMEM. For other experiments, SMC were stimulated with
10% NCS DMEM. The cells were then lysed to obtain total cellular protein with
Protein Lysis Buffer (PLB, see recipe below) containing aprotinin (lOug/ml) and
leupeptin (lOug/ml). After the addition of the PLB, the cells were scraped off of
their growth surface with a Cell Scraper (Life Sciences), transfered to a 1.5 ml tube
and centrifuged at 13,000 rpm for 10 minutes. The supernatant was then collected

and placed in a 1.5 ml Eppendorf tube, snap frozen in liquid nitrogen, and then
stored at -70 C.
The protein was then quantified using a Bradford technique (BioRad Protein
Assay Dye Reagent Concentrate) and colorimetrically analyzed by spectrophotome-
ter or by Microplate Reader (Model 550 from BioRad). The first method involves
the use of plastic cuvettes and the addition of 8 ul of sample, 792 ul of distilled,
deionized water (ddE^O) and 200 ul of BioRad dye. Bovine Serum Albumin (BSA)
standards were prepared in duplicate ranging from 1.2ug/ml to 10 ug/ml. Upon
thorough mixing, the cuvettes were placed into the spectrophotometer to obtain an
absorbency (optical density) reading. This was repeated for all samples and a report
was printed showing the absorbances as compared to the prepared standards. Sample
concentrations were determined and the appropriate protein concentrations (20 ug-
100 ug) were then used for loading the SDS-PAGE gel.
Alternatively, protein concentrations were determined using the Bradford
technique, but absorbances were read using a plate reader. Ninety-six-well plates
were used and four standards were prepared in duplicate. BSA standards range
between 62.5 ug/ml and 500 ug/ml in concentration. The samples were prepared as
1:10 dilutions with 2 ul of protein into 18 ul of ddH20, then divided and added to
200 ul of diluted (1:5) BioRad Dye. The plate reader read the plate and absorbances
were printed. The absorbances were then analyzed and the appropriate protein con-
centrations were used for loading the Western gels.

Immunostain Protocol for Cells
Bromodeoxvuridine (BrdU) Immunocvtochemical Protocol for Cell Staining
Cells were treated with BrdU (10 ug/ml) 24 hours prior to fixing.
BrdU is a thymidine analog which is incorporated into DNA during S phase of the
cell cycle (DNA synthesis). BrdU staining is used to determine the cellular relica-
tion index. The percentage of replicating SMC was determined by counting 500
total cells per condition and determining the percent positive BrdU SMC.
The following steps for cell staining were used with Vector Universal Kit, Vector
DAB kit, Becton Dickinson anti-BrdU (#347580):
1) Wash in IX PBS
2) Denature in 2 N HC1
3) Wash in IX PBS
4) Block in horse serum
5) Add Primary Ab (1:200 dilution anti-BrdU)
6) Wash in 1XPBS
7) Add Secondary Ab
8) Wash in IX PBS
9) Add ABC complex
10) Wash in IX PBS
11) Add DAB
12) Wash in IX PBS
13) Rinse in running ddH20
3x3 min
1 x 20 min
3x3 min
1 x 20 min
1 x 60 min
3x3 min
1 x 30 min
3x3 min
1 x 40 min
3x3 min
1 x 7-10 min
1x3 min
1 x 4 min
Immunofluorescent Stain Protocol
SMC were immunofluorescently stained for phospho-ERK, phospho-FAK, pax-

Antibody (primary)
Rabbit P-ERK 1:100
Rabbit P-FAK 1:100-1:200
Phalloidin 1:40
Mouse Paxillin 1:200
Mouse BrdU 1:200
Antibodv (secondary!
Alexa 488 (FITC) 1:100-1:500
Alexa 594 (Texas Red) 1:200
(both anti-rabbit or anti- mouse conjugated IgG
Table 3.1)
Antibodies used for Immunoflorescence and their dilutions.
illin, BrdU, or actin stress fibers using a rabbit anti-phospho-ERK antibody (New
England Biolabs), a rabbit anti-phospho-FAK antibody (Biosource Laboratories), a
mouse anti-paxillin antibody (Transduction laboratories), a mouse anti-BrdU anti-
body (Becton Dickenson), and Rhodamine-labeled phalloidin (Molecular Probes) for
stress fibers. SMC were fixed with 4% Paraformaldehyde for 10 minutes and then
0.1% Triton X for 5 minutes, both at room temperature. Non-specific binding was
blocked using 1% BSA for 30 minutes. Primary antibodies (See Table 3.1) were
added and SMC were incubated for 2 hours at room temperature or overnight at 4
C. SMC were washed in PBS and incubated with the appropriate fluorescently-con-
jugated secondary antibody for 1 hour. SMC were then washed with PBS and

cover-slipped with an aqueous mounting medium containing DAPI (Becton
Dickenson), used to label all cells. Cells immunofluorescently stained for BrdU were
treated with BrdU for 24 hours prior to fixing. To stain for actin stress fibers, SMC
were fixed as described, then incubated with Rhodamine-conjugated phalloidin for
20 minutes. SMC were then washed in PBS and coverslipped as described above.
The flourescent phalloidin stain is derived from a phallotoxin which is iso-
lated from the deadly Amanita phalloides mushroom. They are bicyclic peptides
which bind to specific sites on Filamentous Actin. This actin binding represents the
stress fibers that are involved in focal contacts. Paxillin stains the paxillin protein
which is involved in the focal contacts and therefore represents their location. The
Phospho-FAK antibody stains the phosphorylated form of FAK and therefore repre-
sents activated FAK. All the other primary antibodies attach to their target protein
demonstrating the localization of the particular protein.
SDS-PAGE (Polyacrylamide Gel Electrophoresis)
SDS-PAGE is a technique used to separate proteins according to molecular
weight. The gels used included (1) Novex 4-12% gradient gels (pre-cast) (2) 8%
Agarose Minigels and (3) Large 8% Protein gels (see gel recipes in Table 3.2 and
3.3 for buffer recipes).
For SDS-PAGE and electrophoretic transfers, Novex brand gels, gel boxes
and transfer apparatuses were most often used. The 4-12% Bis-Tris and 7% Tris-

Stacking gel (3%) Separating gel (8%)
Mini gel / Large gel Mini gel / Large gel
Acryl:Bis 30:08 500 ul / 3.5 ml 3 ml /12 ml
1.5 M Tris pH 8.8 0 2.81 ml / 11.25
* 0.5 M Tris pH 6.8 1.25 ml/7.5 ml 0
10% SDS 50 ul / 300 ul 225 ul/900 ul
dH20 3.2 ml /19.1 ml 5.25 ml/21 ml
TEMED 10 ul / 30 ul 22.5 ul / 90 ul
10% APS 33.3 ul / lOOul 62.5 ul / 250 ul
Table 3.2)
PAGE recipes for mini-gels and large protein gels.
makes 1 Liter
2.42 g Tris
8 g NaCl
IPX Tris-glvcine Transfer
makes 5 Liters
15.1 g Tris
72 g glycine
1 L MeOH
IPX TBS-Tween pH 7.6
3 Liters
72.6 g Tris
24P g NaCl
15 ml Tween
IPX electrode Buffer
makes 4 Liters
12P g Tris
576 g Glycine
4P g SDS
Protein Lvsis Buffer
5P mM Hepes
5P mM NaCl
1 mM Na3V04
1% Triton
IP ug/ml aprotinin, leupeptin
Table 3.3)
Western Blotting Buffer and PAGE Buffer recipes.

Acetate gels contain 10 wells and are 1.0 mm thick. The running buffers used for
electrophoresis were a IX NuPage MES [2(N-morholino) ethane sulfonic acid] SDS
Running buffer for Bis-Tris gels, and Tris-Acetate SDS running buffer were used for
the Tris-Acetate gels. A NuPage Antioxidant was added to the upper chamber run-
ning buffer at a concentration of 500 ul per 200 ml of buffer.
The samples were then prepared by adding 5X Novex NuPage Sample Buffer
and Novex Reducing Agent (1:10) to 20-100 ug of total protein. These protein sam-
ples were always kept on ice during preparation. After preparation, they were then
heated to 100 C for 6 to 10 minutes and quickly centrifuged to collect the sample.
Samples were then placed on ice prior to loading the gel(s). BioRad Kaleidoscope
molecular weight standards (ranging from 7.7 to 216.0 kilodaltons) were used (10-
15 ul). The gels were run at 200 Volts for about 45 minutes or until the visible stan-
dard protein front separated into distinct bands.
SDS-PAGE Transfer
After electrophoresis, proteins were transferred to PVDF membranes
(Amersham) using the NOVEX transfer apparatus. PVDF membranes were first
pre-soaked in 100% methanol for 30 seconds to one minute to ensure proper transfer
onto hydrophobic membranes, and then soaked in the IX Novex Transfer Buffer for
15 minutes. Whatmann paper was cut to size and was soaked in IX transfer buffer
for about 15 minutes. The gels were removed from the plastic casting and were
stacked onto the membrane and surrounded by Whatmann paper. The protein gels
were transferred to the membranes at 30 Volts for 1 to 2 hours. After the transfer
was complete, the protein standards on the membranes were marked with a

hydrophobic pen. The membranes were then rinsed with IX TBS-Tween with
0.05% Tween.
Western Blotting
The blots were blocked for 20-30 minutes with 5% non-fat dry milk in IX
TBS blocking solution. After the block, the blots were rinsed with IX TBS-Tween
and incubated with primary antibody (Ab) in a 1% milk and IX TBS solution. The
blots and Ab solution were placed in a seal-o-meal bag and placed on a shaker for
a 2 hour incubation. The Ab dilutions ranged from 1:100 to 1:5000. The primary
antibodies used are shown in Table 3.4.
After primary Ab incubation, the membranes were rinsed with IX TBS-
buffer for 10-20 minutes and then secondary antibody was added (in the same man-
ner as the primary) for about 45 minutes to 1 hour. This Ab is conjugated with a
Anti-Phospho-Tyrosine (mouse)
Anti-Focal Adhesion Kinase (rabbit)
Anti-Total-ERKs (rabbit)
Anti-Phospho-ERKs (rabbit)
Anti-Phospho-FAK (rabbit)
Anti-B-Actin (mouse)
Anti-PDGF-RB (rabbit)
Anti-Paxillin (mouse)
New England Biolabs and Santa Cruz
Upstate Biotech and Santa Cruz Biolabs
New England Biolabs
New England Biolabs
Upstate Biotechnology
Transduction Laboratories
Table 3.4)
Primary Antibodies used in Western Blotting.

Biotinylated Horseradish Peroxidase enzyme that allows for chemiluminescent
detection. The secondary Abs used were Anti-mouse and Anti-rabbit (Amersham) at
1:10,000-1:20,000. After incubation with the secondary, the blots were washed
three times in IX TBS for 15 minutes each.
Enhanced Chemiluminescence Detection (ECL1 is one of the most commonly used
methods to visualize proteins immobilized on membranes after electrophoretic trans-
fer from SDS-PAGE gels (Clias, Vierra, Elkin and Kichler, vol. 3). It is a non-
radioactive method which is based on the production of and subsequent amplifica-
tion of light by chemical enhancers as a result of enzyme catalysis. Kits used for
ECL detection were the ECL and ECL Plus (Amersham), and the Renaissance sys-
tems (NEN technologies).
Chemiluminescent solutions were mixed according to manufacturers direc-
tions and quickly added to the membranes for 1 -5 minutes depending on the kit that
was used. Excess solution was removed from the blots and the blots were wrapped
in plastic wrap and placed in a cassette for exposure to the x-ray film (Hyperfilm
from Amersham). The film detects the amplification of light from the enzyme-cat-
alyzed reaction.
We used immunoprecipitation in order to verify specific proteins from the
Western blot analysis. For these experiments, total cell proteins (100 ug) were incu-
bated overnight, at 4 C with 10 ug of primary antibody. A 10% Protein A

agarose-PLB solution (100 ul) was added and samples were incubated for an addi-
tional 2 hours. Samples were then centrifuged to collect the Protein A-antibody
pellet. The supernatant was discarded and the pellet washed 3x with 500 ul PLB.
Samples were then resuspended in 5X sample buffer, subjected to SDS-PAGE and
analyzed by western blotting as described above.
Map Kinase Assay for Ras-transfected data
SMC were plated on FN, LN, and Matrigel in SFM or in 10% NCS/DMEM
for 24 hours. SMC were then stimulated with fresh 10% CS or kept in SFM for an
additional 10 minutes, washed 2X with cold PBS and lysed in MAP Kinase lysis
buffer (see below). Samples were collected, centrifuged, and the supernatant trans-
ferred to a clean 1.5 ml tube. Total protein was analyzed as described above.
MAP Kinase Assay
To analyze MAP Kinase activity, 100 ug total protein was immunoprecipitat-
ed with anti-ERK 1 and anti-ERK 2 antibodies as described above.
Immunoprecipitates were assayed in triplicate in 20 ul aliquots. Samples were incu-
bated in 20 ul MAP Kinase mix (below) in the presence and absence of myelin basic
protein (MBP) at 30 C for 15 minutes. The reaction was then stopped with 10 ul
25% TCA. Aliqouts (35 ul) of each reaction were spotted onto p81 filter paper. The
paper was washed 4x in 75 mM H3PO4 then lx in acetone. Filters were dried and
counted for in a scintillator. Total counts per million (cpm) were translated and
data was reported as pmoles ATP utilized per minute per mg total protein.

MAP Kinase lvsis buffer:
Tris 25 mM, pH 7.2
(3-glycerophosphate 50 mM
0.5 % triton X-100
DTT 1 mM
MgCl2 2 mM
Na3V04 100 pM
Add protease inhibitors to the buffer:
aprotinin (10 U/ml)
Leupeptin (0.1 mM)
PMSF final 1 mM
MAP Kinase Mix
(3-GP 0.5 mM
Na3V04 20 mM
MgCl2 1 M
ATP 10 mM
[y32P] ATP
EGF Rec. Pept
IP-20 10 mg/ml
EGTA 200 mM
Table 3.5)
Recipes for MAP Kinase lysis buffer and mix.

Plating SMC on perlecan inhibits
mitogen-induced MAP Kinase activation and cell replication.
Increased activation of the MAP kinases (ERK 1 and 2) is known to be asso-
ciated with cell proliferation. We therefore first studied the activation (i.e. phospho-
rylation) of ERK 1 and ERK 2 when SMC were plated on a perlecan matrix. Using
immunofluorescence and a phospho-ERK specific antibody, we found that mitogen-
stimulated MAPK activity was suppressed by plating SMC on perlecan matrices for
30 minutes (See Figure 4.1). In contrast, we detected abundant levels of phosphory-
lated ERK in SMC plated on fibronectin and collagen type I. DAPI is a nuclear
stain that identifies total cells within the field.
We next plated SMC on fibronectin or perlecan matrices in the presence of
10 mM BrdU. Cells were fixed 24 hours later and immunofluorescently stained for
pERK, BrdU and DAPI. As shown in Figure 4.2, SMC plated on FN demonstrate
high rates of proliferation (measured as the percent of BrdU-positive SMC) and, at
least some of the BrdU positive SMC continue to show high levels of activated ERK
1 and 2. Positive BrdU staining represents the DNA synthesis of replicating cells,
and therefore, indicates growth. In contrast, SMC plated on a perlecan matrix even
in the presence of 10% CS failed to replicate over 24 hours and did not show
detectable phosphorylated ERK 1/2 (i.e. while the nuclear DAPI stain is very clear,

Figure 4.1)
SMC were plated on perlecan (PN), fibronectin (FN), or collagen type I
(Col I) for 30 minutes in the presence of 10% CS. Cells were then
immunofluorescently stained for phosphorylated ERK 1/2 and for DAPI
(to identify total cells).

V <** <+ t
V r 1
Figure 4.2)
SMC were plated on fibronectin in the presence of 10% CS and 10 mM
BrdU for 24 hr. Cells were then immunofluorescently stained for Brdll, for
phosphorylated ERK 1/2, and for DAPI. Arrows indicate an individual SMC
positive for both BrdU and phospho-ERK.

Figure 4.3)
SMC were plated on perlecan in the presence of 10% CS and 10 mM
BrdU for 24 hours and then immunofluorescently stained for BrdU,
P-ERK, and DAPI.

there is no evidence for a positive signal in the BrdU and P-ERK stained fields)
(Figure 4.3).
Plating SMC on Perlecan inhibits
the activation of Focal Adhesion Kinase
SMC were plated on plastic or on intact basement membranes (Matrigel) in
the presence of 10% CS. Total protein was extracted 24 hours later and analyzed by
western blot analysis. Figure 4.4a shows that FAK is constitutively expressed by
Figure 4.4a)
SMC were plated on plastic or on Matrigel in the presence of 10% CS.
Cells were lysed 24 hr later and analyzed by western blot analysis for total
FAK protein (top panel). The filter was stripped and reprobed with a phos-
phosphotyrosine antibody (bottom panel).
Figure 4.4b)
SMC were plated on collagen type I, laminin, collagen type IV, fibronectin
and Matrigel in the presence of 10% CS for 24 hr. Total cell proteins were
immunoprecipitated using a FAK-specific antibody. FAK immunoprecipi-
tates were analyzed by western blot analysis using a phospho-tyrosine anti-



SMC plated on both substrates (top panel) but activation (tyrosine phosphorylation)
is inhibited upon plating SMCs on perlecan-rich basement membranes (Matrigel).
This figure illustrates that the total protein levels are maintained in the cells when
plated on Matrigel, but the FAK protein is not phosphorylated when cells are plated
on Matrigel compared to the cells plated on plastic, which show a signal for activa-
tion of FAK (phosphorylation of tyrosine residues).
Immunoprecipitations were next performed in order to determine if the tyro-
sine phosphorylated pl25 protein was FAK. SMC were plated on Col I, LN, Col IV,
FN, and Matrigel in the presence of 10% CS. FAK immunoprecipitates were ana-
lyzed by western blot analysis for tyrosine phosphorylation. As shown in figure
4.4b, cells plated on Col I, LN, Col IV and FN, show high levels of FAK phosphory-
lation. In contrast, SMCs plated on perlecan-rich basement membranes (Matrigel)
demonstrate low FAK activation.
To determine if perlecan has the same effects on FAK as observed with intact
basement membranes (Matrigel), western blot analysis was performed on total cell
proteins extracted from SMC plated on plastic, firbronectin, or perlecan in the pres-
ence of 10% CS. As shown in Figure 4.5, FAK is activated upon plating SMC on
FN and plastic, but is suppressed by plating the cells on Perlecan matrices. Here
again, we show that the total FAK protein is constant, but the activated levels of
FAK are decreased in these cells.
Lastly, an IP with a FAK antibody that is then probed with a Phospho-tyro-
sine antibody shows that FAK is active in SMC plated on FN, Col I, and Col IV
matrices, but not in SMC plated on perlecan matrices (Figure 4.6). Therefore, we
have demonstrated with numerous techniques, that SMCs plated on perlecan (or a

Figure 4.5)
Phospho-FAK SMC were plated on plas-
tic or on fibronectin or per-
lecan matrices in the pres-
ence of 10% CS Total cell
protein was analyzed by
western blot analysis for
total FAK (bottom panel)
Total FAK and for tyrosine phospho-
rylated FAK (upper panel).
FN Col I Col IV PN
Figure 4.6)
SMC were plated on fibronectin (FN), collagen type I (Col I), collagen type
IV (Col IV) or on perlecan (PN) in the presence of 10% CS. Cells were
then lysed, and total protein immunoprecipitated using a FAK-specific anti-
body. Western blots of FAK immunoprecipitates were then analyzed for
tyrosine phosphorylation using a phospho-tyrosine antibody.

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Constitutively active
Ras-GTP-transfected SMC
How eetaL, 1998,
Renshaw et aL, 1997,1999
Oktay etal., 1999
Figure 4.7)
Schematic illustrating the pathway that is constitutively active when SMC
are transfected with a Ras-GTP (active form). [Despite the presence of
perlecan, Ras-GTP can potentially still cause growth via the ERK-1/2.]
perlecan rich basement membrane) do not exhibit activated FAK, a read out for inte-
grin-mediated signaling.
The growth inhibitory effect of Perlecan is overridden
bv activating the MAP Kinase signaling pathway
When observing Figure 4.7 we are reminded where the Ras protein fits into
the growth stimulatory pathway. To determine if the growth inhibitory effects of
perlecan are acting upstream of the Ras-MAP Kinase pathway, SMCs were stably

Plastic 10% CS
Basement Membrane 10% CS
Figure 4.8)
Empty vector-transfected (vector) or Ras-GTP-transfected (Ras-GTP)
SMC were plated on plastic (blue bars) or on Matrigel (red bars) in the
presence of 10% calf serum.
Left: Total MAP Kinase activity was measured as the ability of ERK 1/2
immunoprecipitates to phosphorylate myelin basic protein.
Right: numbers were determined in triplicate after 5 days in culture.
transfected with a constitutively-activated Ras-GTP construct (Ras-GTP). These
cells were plated on plastic or Matrigel and analyzed for MAP Kinase activity and
for their ability to increase cell number in response to serum stimulation. As shown
in Figure 4.8, empty vector transfected cells showed decreased MAP Kinase activity
when plated on Matrigel, compared to cells plated on plastic, similar to wild type
SMC. In contrast, Ras transfected SMC plated on Matrigel retained high MAP
kinase activity, comparable to that measured from Ras-transfected cells plated on

Next we counted total cell number at Day 0 and Day 5 of empty vector
SMCs and Ras-GTP SMC plated on Plastic and Matrigel in the presence of 10% CS.
Similar to the wild-type SMC, empty vector-transfected SMC increased cell numer
when plated on plastic, but failed to increase cell number when plated on Matrigel
(Figure 4.8). In constrast, Ras-transfected SMC grew prolifically on both plastic and
on Matrigel and showed greater increases in cell number than the empty vector
transfected cells grown on plastic. Therefore, we found that mitogen-activated ERK
1/2 activity and cell replication is not suppressed in Ras-GTP transfected SMC when
they are plated on basement membrane gels (matrigel) (Figure 4.8). These data sug-
gest that high MAP Kinase activity correlates to high cell growth levels.
Finally, we looked at the activity of FAK when the Ras-GTP cells are plated
on the Matrigel compared to plastic using western blot analysis. As shown in Figure
4.9, Ras-GTP SMC plated on basement membranes exhibit high levels of FAK acti-
vation compared to those plated on plastic. Consistant with this, Ras-GTP SMC
remain rounded when plated on plastic, but exhibit increased focal contacts and
spreading when plated on basement membranes. All of these experiments show that
the Ras-GTP transfected cells have increased MAP kinase activity and total cell
growth rates when placed on perlecan-enriched membranes. The growth inhibitory
effect of the basement membrane is therefore overridden by constitutively activating
the Ras-MAP Kinase pathway. Therefore, the inhibitory effects of perlecan must be
upstream of Ras, because otherwise, the constant activity of Ras would have no
effect on growth (cells would remain in a quiescent state as dictated by perlecan).

S 2 Cfl
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Total FAK
Figure 4.9)
Ras-GTP-transfected SMC were plated on plastic or on basement mem-
branes in the presence of 10% CS. Cell lysates were analyzed for FAK
tyrosine phosphorylation (top) and for total FAK protein levels (bottom) using
the western blot technique.
FAKphosphQryiatiQP i? minimaLin.
medial SMC isolated from adult uninjured vessels.
We next tested our hypothesis that perlecan suppresses SMC growth by
inhibiting the FAK signaling pathway in an in vivo system when SMC are extremely
quiescent and surrounded by a perlecan-rich basement membrane. Using western
analysis, we found that tyrosine phosphorylation of FAK is suppressed in SMC from

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Total FAK
Figure 4.10)
SMC were isolated from intact uninjured aortic media and lysed for total
protein (Vivo). Cultured SMC were kept in suspension (Susp) or plated on
plastic (PI), fibronectin (FN), or on perlecan (PN). Cell lysates were ana-
lyzed for total FAK protein (bottom panel) and for activated (Phosho) FAK
(top panel) using western blot analysis.
an intact vessel compared to cultured SMC plated on FN or on plastic (Figure 4.10).
Similarly, SMC in suspension (no attachment to matrix or any substrate) did not
show any activation of FAK and the cells plated on perlecan were only slightly
Since we found that FAK remains dephosphorylated when SMC are plated
SMC plated on Perlecan exhibit
decreased focal contact formation

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on perlecan, but show high levels of phosphorylation when plated on FN and plastic,
we investigated further to better understand the mechanism that drives or hinders
FAK activation. SMC were plated on fibronectin and perlecan for 24 hours and then
immunofluorescently stained with rhodamine-labeled phalloidin to identify actin
stress fiber formation. SMC spread and formed numerous actin stress fibers when
the cells were plated on FN and plastic (Figure 4.11). In contrast, SMC plated on
perlecan remained poorly spread and showed very little stress fiber formation. This
shows that the cells plated on perlecan are not attached and spread in the same man-
Figure 4.11)
SMC were plated on perlecan or fibronectin matrices. Cells were stained
with rhodamine-phalloidin to detect filamentous-actin stress fiber formation
and with DAPI to detect total cell number.

ner as cells plated on fibronectin, an integrin-interacting matrix protein. Similar
results were obtained with SMC plated on other integrin-interacting proteins (Col I,
Col IV, LN; data not shown).
We also double-stained SMC plated on perlecan or on fibronectin with
Phospho-FAK and paxillin to identify focal contacts (Figure 4.12). Paxillin is an
adaptor protein involved in focal contact formation that is activated via phosphoryla-
tion downstream of FAK phosphorylation. We found that after adhesion to perlecan,
Phospho-FAK Paxillin DAPI

Figure 4.12)
SMC were plated on perlecan or fibronectin for six hours, cells were then
fixed and stained immunofluorescently for activated FAK (Phospho-FAK)
and paxillin. The cells were counterstained with DAPI to identify cell
nuclei. Arrows indicate focal contacts containing phosphorylated FAK and

SMC demonstrated poor spreading, the number of focal adhesions was very low and
there was little or no phosphorylated FAK present. In contrast, SMC plated on
fibronecin formed a great number of focal contacts which contained paxillin and
activated (phosphorylated) FAK. We also tested the SMC on other integrin-interact-
ing matrices (i.e., collagen I and IV, laminin) and found showed similar results (not
Plating SMC on Perlecan results in the
inhibition of growth factor receptor activition
In order to understand the effects of the perlecan on growth factor receptors
and growth factor induced signaling pathways, we studied the PDGF (Platelet
Derived Growth Factor) receptor (Figure 4.13). SMC were growth-arrested in
serum-free media (SFM) for 48 hr then plated on fibronectin or on Matrigel in SFM.
Cultures were treated with or without PDGF-BB for 5 minutes prior to lysing. Some
cells were not plated, but were left in suspension and treated with or without PDGF-
BB. SMC plated on Fibronectin showed a large increase in PDGF-Beta receptor
tyrosine phosphorylation in response to PDGF. However, SMC plated on Matrigel
or kept in suspension, exhibited only a slight phosphorylation of the PDGF-Beta
receptor after PDGF treatment. This demonstrates that the perlecan-rich basement
membrane suppresses GF-Receptor activation, thus down regulating GF receptor
signaling pathways as well as integrin signaling pathways.

FN Mat Susp
Figure 4.13)
SMC were growth arrested for 48 hours and then re-plated on either
fibronectin, Matrigel in serum-free media, or left in suspension. The cells
were then stimulated with10ng/ml PDGF-BB for 5 min. Cell lysates were
collected and analyzed for PDGF-RB activation (phosphorylation) using a
phosphotyrosine antibody as well as for total protein (PDGF-RB and B-
Because we know that perlecan is endogenously produced by the SMCs in
the normal uninjured adult artery (Weiser et al., 1996), we wanted to study the cell
growth after the perlecan is removed from the cell culture environment. To study
this question, serum-induced growth was assessed in the presence or absence of a
neutralizing perlecan antibody (Zymed Laboratories). Total cell numbers were
determined after 3 and 5 days of treatment. We found a 3.5 fold increase in
response to serum stimulation in untreated SMC cultures. In contrast, there was a
7.5 fold cell number increase when the perlecan was abolished with the neutralizing
Endogenouslv-produced perlecan
attenuates mitogen-induced SMC growth


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Day 0 Day 3 Day5
Figure 4.14)
Adult rat aortic SMC were plated on plastic in the presence of (red bars) or
absence (red bars) of a neutralizing antibody against perlecan. Total cell
numbers were assessed at days 0, 3 and 5 of serum stimulation.
antibody (Figure 4.14). This suggests that the perlecan produced endogenously by
the cells depresses the replication of cells in culture. Hence, it is likely that perlecan
being produced by the cells, as well as the perlecan in the intact basement membrane
both play a role in the SMC quiescence.

Neointimal formation, due in part to Smooth Muscle Cell (SMC) prolifera-
tion, is a fundamental complication of Vascular Heart Disease. In order to better
understand the cellular mechanisms of this formation and cellular proliferation, the
cells environment and the cellular signals inside the cell must be studied in great
detail. SMC proliferation is a delicate balance of stimulatory and inhibitory extra-
cellular and intracellular influences. While the stimulatory influences, such as
growth factors and proliferative matrices are becoming better understood, the
inhibitory influences that keep the cells from growing are not well known. A better
understanding of the quiescence of a healthy and mature SMC can lead to the design
of potential endogenous therapeutics that could revert a diseased vessel back to its
healthy contractile state. This understanding must come from more specific studies
of the matrix and basement membrane that provide quiescent messages to the cells,
as well as the cell signaling cascade that prevents the proliferation of the cells.
Endogenous heparan sulfate proteoglycan molecules are believed to play a role in
the suppression of vascular SMC replication. Perlecan is the predominent heparan
sulfate-containing proteoglycan found in all vascular basement membranes. It is
about 400-450 kilodlatons in weight and contains 5 domains. The protein core of
Perlecan attaches many heparan sulfate sidechains and in combination, these are
believed to be responsible for the inhibitory signaling pathways that are initiated

when Adult SMC are plated on perlecan or basement membranes (Belknap et al.,
1999, Clowes et al., 1977, 1983, Cook et al., 1993, Guarda et al., 1996, Murdoch et
al., 1994, Pauly et al., 1992, Schwartz et al., 1995).
Previous studies indicate that cell adhesion to the ECM induces integrin clus-
tering and the generation of biochemical signals within the cell which provide the
means for the cell to undergo a change. These signals include the activation of tyro-
sine kinases such as Focal Adhesion Kinase, Src, She, and the ERKs which are ser-
ine/threonine kinases (Alpin and Juliano, 1999, Oktay et al., 1999, Schaller, et al.,
1994, 1999). Because normal cell growth requires stimulation via adhesion to the
ECM, as well as by growth factors, but little is known about how this synergism
works, we began to study Focal Adhesion Kinase as a potential mediator of all
such signals. We also studied the focal contacts that form as a result of integrin acti-
vation and the inactivation/activation of the FAK-induced signaling pathways when
SMC are plated on perlecan compared to other matrices. MAP Kinases, ERK 1 and
ERK 2, were also studied because they are activated prior to cell growth. In an
effort to compare the effects of SMCs being placed on stimulatory EMC compo-
nents, to our inhibitory ECM (perlecan), we used FAK and the ERKs as read-outs
for growth.
The experiments described in this report were designed to explore the quies-
cent nature of the SMCs when undisturbed and most importantly, the involvement of
the protein perlecan in this process. In the first part of this study, we demonstrated
that SMC adhesion to basement membrane and/or perlecan decreases MAP Kinase
activity as shown by immunostain. The phosphorylated form of ERK (MAP
Kinases) is not evident after the cells were plated for 30 minutes on perlecan com-

pared to those that were plated on fibronectin or collagen I. These stains also illus-
trated a decrease in DNA synthesis, shown by decreased BrdU incorporation after 24
hours compared to the cells that were plated on fibronectin. Therefore, as both acti-
vated ERK and DNA synthesis are read-outs for growth, and both of these are
decreased in this instance, we surmise that when cells are surrounded by perlecan,
growth is suppressed.
The second question we asked involved the activation or inhibition of FAK
when SMCs were plated on perlecan matrices. SMC in culture consdtutively
express FAK, but Western blot analysis demonstrates that SMC plated on Matrigel (a
basement membrane rich in perlecan) do not have activated forms of FAK.
However, when the cells are plated on plastic, the FAK is tyrosine phosphorylated.
Similarly, we found that SMC plated on matrices such as collagen I, IV, laminin, and
fibronectin, exhibit high levels of FAK activation compared to cells plated on
Matrigel or perlecan. These other matrices were found to increase the activation of
the integrin-mediated pathway, while basement membranes did not, as demonstrated
by FAK phosyphorylation levels.
In order to better determine where perlecan fit into the pathway(s) of the
growth stimulation, we constitutively activated the MAP Kinase pathway to assess
whether constitutuve activation of a growth pathway would override the growth
inhibitory effects of the basement membrane. A Ras-GTP construct that is a consti-
tutively active form of Ras, was transfected into the cells and stable clones were
selected. The results demonstrated that MAP Kinase activity is still very high on
Matrigel when cells were transfected with the Ras-GTP, and the cell number also
increases greatly on the same matrix. Interestingly, there is a higher cell count for

the Ras transfected cells plated on Matrigel compared to those on plastic. This
could be a result of a positive feedback loop known as "inside-out" signaling, where
the activation of the proliferative pathway could be causing a positive feedback loop
that recruits more integrins and focal contacts to the adhesion sites, causing more
down-stream signaling (Shattil SJ, Ginsberg, MH, 1997). Therefore, this constant
activation of the MAP Kinase pathway via Ras-GTP overrides the inhibitory effect
of the perlecan-rich basement membrane. Hence, the growth inhibitory effect of
perlecan is initiated upstream of Ras and mitogen-activated SMC replication is not
suppressed by basement membrane gels in Ras-GTP-transfected SMC.
In efforts to test the effects of perlecan on cells from the vessel, we studied
the integrin mediated pathway in SMC isolated from an intact normal quiescent
vessel. These cells were taken from adult rat tissue from an uninjured aorta and
immediately lysed. They were not plated in culture and were never attached to a
substratum. When tested by Western blot for activation of FAK, no FAK phosphory-
lation was found. The same cells did not show large levels of FAK activation when
placed on perlecan or when suspended in media (no attachment) either. In contrast,
the Adult SMCs that were plated out on FN and plastic did show high levels of FAK
Because both growth factor and integrin signaling events are necessary for a
proliferative effect, we studied the attachment of these cells by observing the focal
contacts in the SMC when plated on FN, PI and perlecan. We found that the actin
filaments (which represent stress fibers as a part of focal adhesions), were well

formed on the cells plated on fibronectin and other stimulatory matrices. Cells plat-
ed on a perlecan matrix had very weak actin filament staining and therefore, few or
no stress fibers were present. Also, when paxillin and P-FAK were studied via
immunofluorescence for the presence of focal contacts on Fibronectin, we again saw
great numbers of P-FAK activated cells as well as paxillin positive cells. On the
perlecan plated cells, it was apparent that they did not form strong focal contacts as
positive paxillin and P-FAK cells were not obvious. The data based on PDGF-
Receptor activation enabled us to see that when the cells are plated on basement
membranes, or kept in suspension, with addition of growth factor, the PDGF-
Receptor is not activated. On fibronectin, however, the receptor is activated on the
tyrosine residues in response to PDGF stimulation. Perlecan seems to inhibit signal-
ing through the growth factor receptor stimulatory pathways. These data support the
theory that disruption of both focal adhesions and actin skeleton can be induced by
the effects of perlecan or similar basement membranes, which later leads to down-
stream signaling that can inhibit growth.
In order to test the effects of the endogenously produced perlecan on SMC
replication, we used a perlecan neutralizing antibody after serum stimulation and
performed cell counts. We saw a great increase in cell number after 5 days when
endogenous perlecan protein was neutralized by the antibody. This evidence sup-
ports previous studies that indicate perlecan to be endogenously produced. This evi-
dence also indicates that the production of perlecan from the SMCs is as equally
important as the addition of perlecan to the intact basement membrane for the SMC
quiescent phenotype.
In summary, cell proliferation in the vasculature is greatly involved in a num-

ber of vascular diseases, such as atherosclerosis, angina, vasospasms and diabetes
(Lascalzo J et al., 1992). Elucidation of signaling pathways involved in this growth
phenotype is a significant step towards undertanding the opposite or inhibitory and
healthy phenotype of these adult smooth muscle cells. We have shown in this paper
that FAK and the MAP Kinases are vital components in the stimulatory signaling
pathway and are activated when the cells are surrounded by FN or other stimulatory
matrices. The focal contacts formed when plated on stimulatory matrices are also
growth promoting for the SMCs. However, when the cells are not plated on these
matrices, but plated on a membrane that represents the in vivo environment, the
focal contacts do not form, FAK and MAP Kinases are not activated and the cells
essentially do not grow. Hence, we must elucidate more of the signaling surround-
ing the FAK molecule, determine better integrin specificity with the ECM and its
individual components and lastly, determine the exact mechanisms that perlecan uses
in interacting with adult smooth muscle cells.

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