Matrix Mechanics and Cell Contractility in Angiogenesis - Mechanical and Chemical Signaling in Angiogenesis - page 146

3.1 Integrins and the Actin Cytoskeleton Mediate EC
Mechanosensitivity

Early experiments uncovered a mechanical linkage between the nucleus and cell-
surface receptors, whereby exerting exogenous force on integrins reoriented cyto-
skeletal filaments, distorted the nucleus, and redistributed nucleoli within ECs [ 45 ].
This work suggested that integrin engagement was related to processes governed by
the cellular nuclear machinery, and in fact integrin binding directly activates the
expression of the immediate-early genes c-fos, c-myc, and c-jun that regulate cell
growth [ 46 ]. In addition, integrins ligated by RGD-coated magnetic beads and
twisted with a magnetic stimulator increase endothelin-1 (a vasoconstrictor) gene
expression, a response blocked by disrupting the actin cytoskeleton [ 47 ]. These
findings suggest that mechanical forces, as sensed by integrins, may play an active
role in mediating EC transcription-regulated responses governing angiogenic
processes.
Force transmission throughout the cell is largely facilitated by the actin cyto-
skeleton. Experiments disrupting cell-substrate adhesion and inducing cell
retraction indicate that the cytoskeleton is prestressed, and that the actin cyto-
skeleton is the primary stress-bearing member in ECs [ 48 ]. Cell stiffness increases
when cells are rapidly strained suggesting time-dependent mechanical properties.
Indeed actin stress fibers behave as viscoelastic cables tensed by myosin motors
where actin cytoskeletal prestress is largely determined by myosin light chain
kinase [ 49 ] (Fig. 3 a). Furthermore, a separate study using AFM nanoindentation
characterized the mechanical properties of individual stress fibers in living cells. It
was found that actomyosin contractility regulates the mechanical properties of
stress fibers [ 50 ]. Stress fiber stiffness can be altered by pharmacological pertur-
bation of contractility (e.g. blebbistatin decreased stiffness while calyculin A
increased stiffness). While the baseline mechanical properties of stress fibers are
approximately linear, they become heterogeneous with increasing cellular con-
tractility. These data suggest that the mechanical microenvironment of ECs alters
the mechanical properties of stress fibers within the cell via actomyosin

contractility.
ECs exhibit viscoelastic mechanical properties that are sensitive to changes in
cell spreading. When grown on substrates of increasing fibronectin ligand density,
ECs exhibit increases in spreading, cytoskeletal stiffness, and apparent viscosity,
[ 51 ]. Separate studies using magnetic pulling cytometry determined how cells
adapt to changes in mechanical forces applied at integrins [ 52 ]. Cells exhibit a
four-phase response: immediate viscoelastic; early strengthening response to short
pulse forces (both prevented by inhibiting Rho); robust strengthening after pro-
longed force; large-scale repositioning after prolonged force application. These
responses are prevented by inhibiting Rho, blocking mechanosensitive ion chan-
nels, or inhibiting Src tyrosine kinase. Separate work indicates that treatment of
ECs with VEGF increase reduces the elasticity of the cytoplasm, a responses that
is dependent on ROCK [ 53 ]. These findings indicate that ECs have robust