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Statement of Problems

Flexible ECM facilitates EC traction
force generation in angiogenesis

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            Although angiogenic factors initiate neovascularization,
the phenotypic fate of ECs is regulated by the tissue microenvironment.  For example, stimulation with fibronectin
growth factor (FGF), a soluble endothelial mitogen, can either induce growth,
differentiation, involution, or quiescence of ECs depending on the mechanical properties
of the ECM (Folkman & Ingber, 1987). 
In the study “Mechanochemical Switching between Growth and
Differentiation during Fibroblast Growth Factor-stimulated Angiogenesis In
Vitro: Role of Extracellular Matrix,” Inger and Folkman aimed to discern the
tension-dependent interplay between ECs and the ECM in response to
FGF-stimulated angiogenesis.  To
accomplish this, time-lapse cinematography was used to study various in vitro models of spontaneous EC
angiogenesis.  On rigid culture dishes,
adhesive matrix tendrils accumulated and applied tension to substrate contact
points, resulting in multicellular retraction and elevation of the ECM web into
culture media.  The suspended ECM web
functioned as a flexible attachment that deformed under cell-generated tensile
forces and permitted capillary tube formation, whereas ECs that remained
adhered to the rigid culture dish did not form tubes.  These results are consistent with previous
studies of angiogenesis on tissue culture plastic, which all required release
from contact with the rigid substrate for capillary tubes to form (Folkman
& Haudenschild, 1980; Maciag et al., 1982; Feder et al., 1983; Madri &
Williams, 1983).  Conversely, ECs
cultured on flexible ECM gels spontaneously formed capillaries without release
from the underlying substrate, with tube formation occurring after days
compared to weeks on tissue culture plastic (Nicosia et al., 1982; Madri &
Williams, 1983; Montesano et al., 1983; Schor et al., 1983; Kubota et al.,
1988).  Taken together, the data supports
the notion that malleab le ECM promotes cell-generated tension and EC tube

            To further test this hypothesis, nonadhesive dishes were
coated with different densities of fibronectin (FN), an ECM glycoprotein that
binds integrins, to yield substrates of varying adhesiveness.  Capillary ECs were then cultured on the
FN-coated dishes and stimulated with FGF. 
Serum (containing fibronectin and vitronectin) was excluded from cell
culture media to limit cell attachment to cell-substrate interactions.  On dishes coated with low densities of FN
(<100 ng/cm2), an involuted EC phenotype was observed, with cell rounding, detachment, and loss of viability.  Intermediate FN coatings (100-500 ng/cm2) promoted cell spreading to some degree, but cell-generated traction forces limited cell extension and growth.  With these coating densities, a differentiated EC phenotype occurred, as characterized by multicellular retraction and formation of branching tubular networks from preliminary cellular cords.  In contrast to moderately adhesive ECM, high FN coating densities (>500 ng/cm2) promoted
extensive cell spreading and growth, with cell spreading increasing as a
function of FN coating density.  However,
no observable tube formation occurred at these concentrations.  Adhesive forces could be overcome on densely
coated FN dishes by plating cells at a higher density, resulting in increased
cell-generated traction force and capillary tube formation.  These results were similarly observed for
dishes coated with varying densities of type IV collagen or gelatin, other ECM
components.  Interestingly, under
differentiating conditions, cell growth was suppressed, despite stimulation
with a potent endothelial mitogen.  This
suggests that phenotypic switches may result from tension-dependent changes in
cell signaling pathways that affect sensitivity to morphogenic factors.  Thus, mechanical properties of the ECM, as
determined by its adhesivity and rigidity, may be just as important as chemical
properties in governing morphogenesis. 
This study presents early evidence that a malleable ECM facilitates traction
force during angiogenesis, although the mechanism of differentiation is not


GTPase Rho mediates mechanosensing of
the ECM

            Tumor blood vessels exhibit marked structural and
functional abnormalities compared to normal vasculature.  They are dynamically changing, irregularly
shaped, and contain abnormal pericyte and basement membranes (Baluk et al.,
2005).  While VEGF-A, a soluble
angiogenic growth factor, has been historically targeted to normalize
vasculature, its effects are transient. 
Since force is transmitted by the ECM through integrin receptors, an
understanding of how tumor vessel cells respond to mechanical stimuli during
angiogenesis may be necessary to effectively treat cancer (Bershadsky et al.,
2003).  In the study “Tumor-derived
endothelial cells exhibit aberrant Rho-mediated mechanosensing and abnormal
angiogenesis in vitro,” Ghosh et al. attempted to elucidate the mechanisms
responsible for the formation of structural abnormalities in tumor
vessels.  They hypothesized that cancer
vasculature results from aberrant mechanosensing mechanisms in tumor capillary
ECs.  To test how ECs respond to physical
cues in their microenvironment, normal and tumor capillary ECs were cultured on
FN-coated silicon substrates and exposed to 10% uniaxial cyclical strain.  Compared to 90% of normal ECs, only 60% of tumor
ECs reoriented their longest axis and actin stress fibers perpendicular to the
direction of applied strain, as visualized using fluorescent microscopy and
staining with Alexa Fluor-488 Phalloidin, respectively.  Since cell shape is influenced by the ability
of ECM to resist cell-generated traction forces, cell spreading was measured as
an indirect indication of ECM rigidity sensing by culturing normal and tumor
ECs on gelatin gels of varying elasticity (citation).  On
soft substrates, normal and tumor ECs exhibited similar shape and size.  However, as substrate stiffness increased, normal
ECs became elongated and thin, while tumor ECs maintained their polygonal shape
and spread to a much greater extent (in terms of projected cell area).  These results clearly indicate that
mechanosensitivity is altered in tumor cells compared to normal cells. 

test whether the aberrant mechanosensitivity of tumor ECs influences
angiogenesis, normal and tumor ECs were cultured on two-dimensional
thrombin-crosslinked fibrin gels at different plating densities.  At low plating densities, normal ECs were
quiescent, whereas tumor ECs formed extensive capillary networks.  In contrast, at high plating densities,
normal cells exhibited capillary formation, whereas tumor cells experienced
multicellular retraction, disruption of the tubular network, and clumping.  This was similarly observed with cells
cultured within three-dimensional fibrin gels, instead of on top.  Normal cell behavior is consistent with the
results of the previous study, in that higher cell densities increase
cell-generated traction force and resulting capillary tube formation (Ingber et
al., 1989).  To confirm that tumor ECs
are indeed more contractile that normal ECs, cells were cultured on thin,
FN-coated polyacrylamide gels containing fluorescent nanobeads and measured
with traction force microscopy.  Compared
to normal ECs, tumor ECs displayed larger regions of stress and applied greater
traction force to their underlying substrates. 
This supports the hypothesis that tension-dependent effects on FAs and
actin stress fibers contribute to aberrant adhesion and spreading observed in
tumor ECs.

            Regulation of cytoskeletal tension generation and focal
adhesion formation has been shown to occur by the small GTPase Rho through its
downstream effector, Rho-associated kinase (ROCK), which mediates phosphorylation
of myosin light chain (Amano et al., 1997). 
Rhotekin pull-down assays and Cyclex Rho-kinase assays were respectively
used to measure Rho and ROCK activity in normal and tumor ECs.  Under normal growth conditions, tumor ECs
exhibited elevated baseline Rho and ROCK levels.  When subjected to 10% uniaxial cyclic stretch
on FN-coated silicon substrates, Rho activity increased in normal ECs, but
remained unchanged in tumor ECs.  This
implies that Rho requires stretch-activation in normal cells but is inherently
activated in tumor cells.  In a prior
study by Tzima et al., transient inhibition of Rho was necessary to relieve
cytoskeletal tension in order for cells to realign in response to shear stress.  For this reason, ECs were treated with the
ROCK inhibitor Y27632 to determine if lowering baseline level of cytoskeletal
tension in tumor ECs could restore normal cell function under uniaxial cyclic
strain.  This resulted in perpendicular
reorientation of actin stress fibers to a greater extent in tumor ECs compared
to untreated tumor ECs (83% versus 60%). 
Additionally, treatment with Y27632 prevented clumping of tumor ECs at
high plating density and instead, promoted capillary tube formation.  This data therefore indicates that the Rho-mediated
mechanosensing plays a critical role in EC response to physical cues from the
ECM, and that aberrant mechanosensing may contribute to abnormal tumor angiogenesis.

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