Cyclic tensile strain triggers a sequence of autocrine and
paracrine signaling to regulate angiogenic sprouting in
human vascular cells
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Citation
Yung, Yu Ching et al. “Cyclic tensile strain triggers a sequence of
autocrine and paracrine signaling to regulate angiogenic
sprouting in human vascular cells.” Proceedings of the National
Academy of Sciences 106.36 (2009): 15279-15284. © 2009
National Academy of Sciences
As Published
http://dx.doi.org/10.1073/pnas.0905891106
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United States National Academy of Sciences
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Detailed Terms
Cyclic tensile strain triggers a sequence of autocrine
and paracrine signaling to regulate angiogenic
sprouting in human vascular cells
Yu Ching Yunga,b, Jeiwook Chaec, Markus J. Buehlerd, Craig P. Hunterc, and David J. Mooneya,e,1
aHarvard
School of Engineering and Applied Sciences and cDepartment of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138;
of Chemical Engineering, University of Michigan, Ann Arbor, MI 48109; dLaboratory for Atomistic and Molecular Mechanics, Department
of Civil and Environmental Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA, 02139; and eWyss Institute
for Biologically Inspired Engineering, Cambridge, MA 02138
bDepartment
Edited by Robert Langer, Massachusetts Institute of Technology, Cambridge, MA, and approved June 30, 2009 (received for review May 29, 2009)
angiogenesis 兩 angiopoietin-2 兩 endothelial cells 兩 shRNA 兩 strain gradient
A
ngiogenesis requires an orchestrated series of cell activities
in a specific spatial and temporal sequence (1). Significant
research in this field has focused on documenting cell response
to exogenous biochemical cues, and a variety of growth factors
have been identified to have key roles in angiogenesis (2).
Among these biochemical cues, VEGF has been identified as a
potent factor during the early stages of angiogenesis, activating
migration, and sprout formation (3). Angiopoietin (Ang)-1, a
cytokine that mediates the interactions between endothelial cells
(ECs) and smooth muscle cells (SMCs), and Ang-2, an early
angiogenic factor that inversely acts to disrupt and dissociate
these bonds, are ligands expressed by vascular cells that competitively bind to the membrane receptor Tie-2, and act synergistically with VEGF to regulate angiogenesis (4). PDGF-␤␤, a
chemotactant released by ECs, is a late stage cytokine that
recruits SMCs to stabilize the nascent EC sprouts (5).
Understanding of the effects of these soluble factors alone is,
however, likely insufficient to fully understand the angiogenic
process. Physiologically, ECs and SMCs are exposed to cyclic
tensile strain resulting from blood hemodynamic forces, a cue
important to vessel adaptation (6). Mechanical signals regulate
the function of both cell types in vitro, including the alteration
of EC proliferation (7, 8), alignment (9, 10), migration (11, 12),
and in vitro sprout formations (13, 14), likely through activating
various intracellular signaling pathways (15–21). Similarly, physiologically relevant hemodynamic forces have demonstrated to
modulate SM phenotype (22), migration (23), and intracellular
signaling (24). Altogether, these past studies suggest that the
www.pnas.org兾cgi兾doi兾10.1073兾pnas.0905891106
angiogenic process is governed by an interplay between chemical
and mechanical signals.
We hypothesized that cyclic tensile strain may provide a
singular cue to regulate vascular cells by triggering EC secretion
of angiogenic factors that mediate multiple stages in the angiogenic process. Human umbilical vein (HUV)ECs and human
aortic (HA)SMCs were used here as model cell types representing the vascular endothelium and stabilizing supportive layer,
respectively. Vascular cells were cultured in 2D directly on
elastomeric poly(dimethylsiloxane) (PDMS) substrates and in
fibrin 3D cultures. In vitro sprouting was used as a model for the
initial stages of angiogenesis, as previously described (25–28).
Vascular cell cultures were exposed to cyclic tensile strain with
an amplitude of 7%, representative of that experienced by
vascular cells in large vessels, and also in the same range as
previous calculations of capillary wall strain (29, 30). Cyclic
tensile strain was demonstrated to alter vascular cell phenotype
and the secretion of Ang-2 and PDGF by EC. Altered Ang-2
secretion mediated changes in early angiogenic processes, EC
migration, and in vitro capillary formation, whereas strainenhanced secretion of PDGF provided a directional cue for SMC
recruitment toward EC colonies.
Results
Cyclic Tensile Strain Regulation of Vascular Cell Phenotype, Angiogenic Factor Secretion, and Migration. The previously reported
effects of cyclic strain on EC migration and in vitro sprout
formation were first confirmed. Cyclic tensile strain (7%, 1 Hz)
enhanced, by 1.6-fold, the directional migration of HUVECs in
2D culture (Fig. S1 A), as expected (31). Two days of cyclic
tensile strain also enhanced sprout formation by HUVECs in
fibrin gels by 4-fold, compared with static culture (Fig. S1B),
again in agreement with earlier investigations (13). Addition of
recombinant human VEGF-165, a known stimulant to capillary
formation (3, 5), also increased sprout formation both under
static and strained conditions (Fig. S1B), confirming the expected biological responsiveness of the cells used in these studies.
The effect of varying the magnitude of cyclic tensile strain was
first assessed by analyzing the fraction of cells that migrated out
from the original cell culture region, the average cell velocity,
and directionality of migratory cells. Cyclic tensile strain enhanced the fraction of HUVECs that migrated out of the original
culture region by 2-fold when an amplitude of 6% cyclic strain
Author contributions: Y.C.Y., M.J.B., and D.J.M. designed research; Y.C.Y. performed
research; J.C. and C.P.H. contributed new reagents/analytic tools; Y.C.Y., M.J.B., and D.J.M.
analyzed data; and Y.C.Y. and D.J.M. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1To
whom correspondence should be addressed. E-mail: mooneyd@seas.harvard.edu.
This article contains supporting information online at www.pnas.org/cgi/content/full/
0905891106/DCSupplemental.
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Mechanical signals regulate blood vessel development in vivo, and
have been demonstrated to regulate signal transduction of endothelial cell (EC) and smooth muscle cell (SMC) phenotype in vitro.
However, it is unclear how the complex process of angiogenesis,
which involves multiple cell types and growth factors that act in a
spatiotemporally regulated manner, is triggered by a mechanical
input. Here, we describe a mechanism for modulating vascular cells
during sequential stages of an in vitro model of early angiogenesis
by applying cyclic tensile strain. Cyclic strain of human umbilical
vein (HUV)ECs up-regulated the secretion of angiopoietin (Ang)-2
and PDGF-␤␤, and enhanced endothelial migration and sprout
formation, whereas effects were eliminated with shRNA knockdown of endogenous Ang-2. Applying strain to colonies of HUVEC,
cocultured on the same micropatterned substrate with nonstrained
human aortic (HA)SMCs, led to a directed migration of the HASMC
toward migrating HUVECs, with diminished recruitment when
PDGF receptors were neutralized. These results demonstrate that
a singular mechanical cue (cyclic tensile strain) can trigger a cascade
of autocrine and paracrine signaling events between ECs and SMCs
critical to the angiogenic process.
A
B
C
D
E
F
G
H
Fig. 1. Cyclic tensile strain up-regulated secretion of angiogenic factors by vascular cells in a temporal manner. (A–D) Secretion of PDGF, Ang-2, Ang-1, and
VEGF by HUVECs and HASMCs was quantified after exposure to 5 days of cyclic strain. Protein levels were quantified using enzyme immunoassays and values
(n ⫽ 3) were normalized to total cell number per well. (E and F) Expression profiles of HUVECs secretion of Ang-2 and PDGF in response to 14 days of cyclic strain.
Values represent normalized levels (n ⫽ 3) under the cyclic strain condition normalized to levels secreted under static (no strain) conditions. (G) Cyclic strain effect
on gene expression of Ang-2 and Tie-2 mRNA levels, after a duration of 5 days, was quantified by real-time RT -PCR (n ⫽ 6). Expression levels, with and without
cyclic strain application, were normalized to GAPDH levels and the ratio of strained to nonstrained levels are presented in the graph. (H) Expression of PDGF-R
was enhanced after 48 h of cyclic strain application to HASMCs, as documented using immunohistochemistry directed to PDGF-R (green fluorescence). *, P ⬍ 0.05;
**, P ⬍ 0.005.
was applied, whereas at an amplitude of 13% cyclic strain, the
fraction of migrating cells was enhanced ⬇4-fold (Fig. S1C). The
average velocity of HUVEC migration at 13% cyclic strain was
15-fold higher, as compared with the static condition (Fig. S1D),
resulting in an average velocity of 15 ␮m/h. Further, the migration of HUVECs exhibited a clear directionality, perpendicular
to the direction of strain application (Fig. S1E). Similarly, 2 days
of cyclic tensile strain led to a 3-fold enhancement in the number
of migrating HASMCs at 13% strain, as compared with the
nonstrained condition, but little effect was noted at 6% strain
amplitude (Fig. S1F). The average velocity of migrating
HASMCs, at 13% strain, was ⬇6-fold higher than cells in static
15280 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0905891106
culture, but was notably slower than HUVEC migration rates
(Fig. S1G). Strikingly, and in contrast to HUVECs, whereas
HASMCs migration was increased with cyclic strain, the cells did
not demonstrate directional migration under these conditions
(Fig. S1H).
To examine whether cyclic strain up-regulated the expression
of genes involved in angiogenesis, the levels of angiogenic
proteins secreted by the vascular cells were quantified over a
5-day time-course of cyclic strain. Cyclic strain of HUVECs led
to a 4.8-fold up-regulation of Ang-2 (Fig. 1A), and a 5-fold
up-regulation in the secretion of PDGF-␤␤ (Fig. 1B). In contrast,
cyclic strain of HASMCs resulted only in a slight enhancement
Yung et al.
A
B
C
D
Fig. 2. RNAi was used to determine the role of Ang-2 in HUVEC response to cyclic strain. (A) Effectiveness of shRNA knockdown of endogenous Ang-2 secretion
by HUVECs transfected with shRNA to Ang-2, a control shRNA (scrambled sequence), and control untreated cells. Ang-2 was quantified using enzyme
immunoassays, daily (n ⫽ 5) over 4 days. Values represent mass of protein secreted, normalized to total cell number per well. (B) Level of static HUVEC migration,
over 24 h, across transwell inserts, normalized to untreated control cells, with either shRNA to Ang-2 or scrambled shRNA control (scrambled treatment) (n ⫽
4). (C) Level of HUVEC migration in response to 48 h cyclic strain on PDMS. Values represent number of cells under cyclic strain that migrated out of original
confined circular region, d ⫽ 2 mm, normalized to static, nonstrained conditions (n ⫽ 3). (D) Formation of sprouts under application of strain was quantified using
HUVECs with and without Ang-2 shRNA treatment, in culture medium with or without added VEGF. Values (n ⫽ 3) are normalized to a nonstrained, no growth
factors control. *, P ⬍ 0.05; **, P ⬍ 0.005.
of Ang-1 (Fig. 1C), whereas secretion of VEGF did not appear
to be effected by strain (Fig. 1D). In both strained and nonstrained conditions, the secretion of PDGF-␤␤ and Ang-2 by
HASMC, and Ang-1 and VEGF by HUVECS, respectively, was
minimal. The time course of up-regulation of PDGF-␤␤ and
Ang-2 secretion by HUVECs was next investigated. Ang-2
expression was increased 3-fold by strain at day 1, and then slowly
subsided over the ongoing 13 days to control levels (Fig. 1E).
PDGF-␤␤ secretion, in contrast, did not rise until 2 days of cyclic
stretch, and then quickly returned to baseline control levels (Fig.
1F). Because minimal effects of cyclic strain on angiogenic factor
secretion by HASMC were noted, all subsequent studies of
factor secretion focused on HUVECs. The gene expression
levels of Ang-2, and its receptor (Tie-2) in HUVECs, was
analyzed using real-time RT-PCR, and application of cyclic
strain resulted in 1.5- and 2-fold increases in mRNA levels for
Ang-2 and Tie-2, respectively (Fig. 1G). Last, cyclic tensile strain
was also found to up-regulate surface availability of PDGF-␤␤
receptors (R) on HASMCs (Fig. 1H).
increase in Ang-2 expression in ECs that result from cyclic strain
is sufficient to increase the angiogenic activity of these cells.
To test whether cyclic strain-induced up-regulation of Ang-2
was causative for the strain induced increase in EC migration and
sprouting, RNAi was used to knockdown the endogenous expression of Ang-2 in HUVECs. HUVECs were transfected with
a plasmid that was designed and constructed to release a 63-mer
shRNA that binds specifically to the intracellular mRNA of
Ang-2 and blocks the translation of this protein. Examination
of Ang-2 secretion by cells positively transfected with shRNA
(Ang-2) confirmed a dramatic inhibition of Ang-2 expression for
4 days after treatment (Fig. 2A). The baseline (no cyclic strain)
migration of ECs with shRNA (Ang-2), was decreased by
⬇1.6-fold (Fig. 2B), whereas cells subjected to strain exhibited
a 2-fold decrease in migration with shRNA treatment (Fig. 2C).
Inhibiting Ang-2 also resulted in a 2.2-fold decrease in sprouting
with exposure to cyclic strain, in both the absence and presence
of exogenous VEGF in the culture medium (Fig. 2D).
whether the levels of altered angiogenic factors resulting from
cyclic strain were capable of altering EC phenotype, HUVECs
in fibrin gels were exposed to exogenous recombinant human
Ang-2, Ang-1, PDGF, and VEGF, at levels corresponding to
those produced by cells under strained conditions. Ang-2 and
VEGF enhanced the formation of sprouts (Fig. S2 A and B),
whereas PDGF-␤␤ (Fig. S2C) and Ang-1 (Fig. S2D) had no
discernible effects. The effects of these factors on HUVEC
migration across porous transwell membranes was next examined, and VEGF was found to enhance HUVEC migration (Fig.
S2E), concurring with previous descriptions of the effects of this
cytokine (32). Ang-2 similarly enhanced HUVEC migration,
whereas Ang-1 had no effect (Fig. S2E). Altogether, these data
suggests new roles for Ang-2 in EC biology, and indicate the
Yung et al.
next series of studies examined whether strain induced change in
EC cytokine expression could alter HASMC function, and in
particular whether PDGF-␤␤ could serve to recruit SMCs to
forming vessels. In the first experiment, HASMCs were exposed
to increasing doses of exogenous recombinant human PDGF-␤␤,
and migration was found to increase with PDGF-␤␤ concentration (Fig. 3A). Next, conditioned media was taken from strained
ECs and added to HASMC culture, and this enhanced HASMC
migration by ⬇2-fold, as compared with conditioned media
taken from cells under nonstrained cultures. To determine
whether the enhanced SM migration was a direct result of the
strain-mediated increased PDGF-␤␤ levels, we neutralized
PDGF-R on HASMCs and reassessed their response to conditioned media taken from strained ECs. Neutralizing PDGF-Rs
decreased migration by 45% (Fig. 3B). Next, to determine
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Cyclic Strain Directs SMC Recruitment Toward Migrating ECs. The
Role of Ang-2 in EC Migration and Sprout Formation. To determine
A
B
C
Fig. 3. PDGF enhanced HASMC migration and recruitment. (A) Exogenous application of increasing concentrations of rhPDGF enhanced SM migration across
transwells inserts after 24 h in static culture (n ⫽ 4). (B) Application of conditioned medium from ECs cultured under static or cyclic strain (strain) conditions, with
and without addition of neutralizing antibodies to PDGF-R, was added to HASMCs and their migration was quantified (n ⫽ 4). (C) SM migration was enhanced
when a depot source of PDGF: rhPDGF or conditioned media (CM) from strained ECs was placed in a depot located 2 mm away from the HASMC colon. Migration
results are presented as the fraction of cells that migrated out of the original culture area as compared with a control (no growth factor) condition (n ⫽ 4).
*, P ⬍ 0.05; **, P ⬍ 0.005.
whether PDGF-␤␤ has the ability to direct HASMC migration
over significant distances, a depot of either recombinant human
(rh)PDGF or a depot of conditioned media from strained cells
was placed 2,000 ␮m away from patterned cultures of HASMC.
The migration of HASMCs toward both depots increased by 2and 2.25-fold, respectively (Fig. 3C), as compared a blank depot
with no stimulants.
Finally, the ability of cyclic strain of ECs to induce not only
directed EC migration, but also recruitment of nonstrained
SMCs was assessed. Micropatterned cultures were developed
(Fig. 4A), in which ECs were subjected to cyclic strain or
maintained under static culture, whereas adjacent colonies of
HASMC were subjected to minimal strain. Culture of SMCs
adjacent to nonstrained ECs led to a 2-fold increase in SMC
migration (Fig. 4B). Strikingly, when SMCs were cocultured
adjacent to strained HUVECs, the migration of HASMCs was
enhanced by 5-fold (Fig. 4B), as compared with a negative
control (SMCs with no coculture, no strain). There was no
notable directionality of HASMC migration when cultured
adjacent to nonstrained HUVEC colonies. However, strained
HUVECs not only increased HASMC migration, but also
provided cues to direct the migration of HASMCs toward the
migrating HUVEC colony (2-fold increase in directional migration) (Fig. 4 C and D).
Discussion
The results of these studies demonstrate that cyclic strain can
activate endogenous biochemical cues to direct vascular cells
through sequential stages important to the angiogenic process.
The angiogenic phenotype of ECs, characterized here by directed cell migration and in vitro sprout formation, was enhanced by recombinant Ang-2 and in response to cyclic uniaxial
strain. Cyclic strain increased the early expression of both Ang-2
and its receptor, Tie-2, in ECs, and this increased Ang-2
expression was found to mediate the cyclic strain induced
alterations in EC angiogenic phenotype. Cyclic strain enhanced,
at a later time point than Ang-2, the secretion of PDGF-␤␤ by
ECs, and the PDGF-␤␤ modulated the directional migration of
15282 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0905891106
HASMC toward EC colonies (Fig. S3). Although past studies
have largely focused on vascular remodeling of precapillary
structures (e.g., arterioles) in response to altered load, these
results suggest that the capillaries themselves may also undergo
significant remodeling in response to mechanical cues. Breakdown or structural damage to capillary walls resulting from
disease may provide one situation in which vascular cells experience altered loading (33); exercise may also provide this type
of stimulus, and previous studies have noted alterations in
capillary densities with exercise (34). Previously calculated strain
levels in capillary walls during systolic contraction and stable
diastolic arrest range between 0 to 22% (30, 35), supporting the
physiologic relevance of the 7% strain amplitude used in of our
studies (29). Other investigations have used similar strain levels
to study the effects of mechanical cue effects on ECs in vitro,
specifically in the context of angiogenesis (36–38).
Cyclic strain up-regulated EC secretion of angiogenic cytokines, specifically, PDGF-␤␤ and Ang-2. Previous studies reported that cyclic strain enhanced the expression of PDGF-R
(12, 39), and shear stress enhanced gene expression of PDGF-␤␤
(40, 41) and Tie-2 (42). However, up-regulation of PDGF-␤␤
and Ang-2 in response to cyclic strain has, to our knowledge, not
been previously documented. Interestingly, VEGF, a potent
factor in angiogenic activation, was not affected by strain,
although regulation of this cytokine is governed by other local
cues (43). The temporal profile of increased angiogenic cytokine
secretion by ECs in response to cyclic strain was striking, because
Ang-2, a factor important to the initiation of angiogenesis (44),
was up-regulated early, followed by later expression of PDGF␤␤, which plays important roles in later stages of angiogenesis
(44, 45). These data suggests that cyclic strain modulates angiogenesis by altering the balance of angiogenic factors, and by
temporally mediating the up-regulation of factors driving activation versus those promoting subsequent vessel stabilization.
Ang-2 was found in this study, even in the absence of
mechanical stimulation, to enhance sprout formation and migration of ECs. The role of angiopoietins in vascular development has been the subject of active investigation, and until
Yung et al.
A
C
B
D
Fig. 4. HASMC migration is enhanced and directed by cyclically strained HUVECs. (A) An array of cell colonies (either of HASMCs alone or cocultured with
HUVECS) were cultured on PDMS well surfaces that presented a surface strain gradient (where under application of strain, white region experienced no strain,
and gray regions 8% strain). HASMCs were seeded in the central column (white), whereas HUVECs (under cocultured conditions) were seeded in colonies to the
right or left regions (gray). (B) Migration of statically cultured HASMC, alone and in response to coculturing with static and cyclically strained HUVECs, after 48 h
(n ⫽ 4). (C) Mosaic of 30⫹ images (at 100⫻) illustrating directed migration of HASMCs toward cyclically strained HUVECs. (D) Effect of HUVEC cocultures, both
under static and strained conditions, on directing migration of HASMC colonies based on a hemisphere partition (n ⫽ 4).
Yung et al.
and indirect responses (e.g., secondary to EC-induced alterations in gene expression resulting from cyclic strain). The
finding that HASMCs cultured under static conditions can be
actively and directionally recruited to strain-stimulated ECs
supports the potential physiologic relevance of these findings.
The sequential regulation by cyclic tensile strain of early and
late stages of vascular remodeling represents a previously unrecognized and potentially critical role for mechanical signaling
in angiogenesis. Reciprocal signaling of EC-induced SMC recruitment via PDGF has been examined in vivo (63, 64), but not
with events activated by external mechanical stimuli. More
broadly, the findings of this study provide a specific example of
how localized mechanical signals can be translated into biochemical cues (65) capable of signaling over physiologic relevant
distances. This coupled mechanism may provide multiple points
to intervene and regulate the angiogenic process, and may also
improve the current understanding of various vascular diseases.
Materials and Methods
For cell culture, HUVECs (Cambrex) and human HASMCs (Cambrex) were
cultured at 37 °C, 5% CO2 in endothelial growth medium (EGM)-2 and smooth
muscle cell growth medium (SmGm)-2, respectively (Cambrex), containing 2%
FBS. HUVECs were used between passages 3 and 6, and HASMCs were used
between passages 3 and 7. Cocultures of HUVECs and HASMCs were maintained in culture medium constituted of 1:1, EGM-2 and SmGm-2.
Detailed information on quantification of migration in response to chemotactic gradients, quantification of migration in response to cyclic strain, in
vitro angiogenesis, sprouting assay, quantification of angiogenic protein
secretion, real-time RT-PCR, vector construction and synthesis, plasmid transfection and FACS, creation of an array of isolated cell cultures, and quantification of Vascular Cell Migration in response to cyclic tensile strain are
included in the SI Materials and Methods section.
ACKNOWLEDGMENTS. This work was supported by the National Science Foundation Materials Research Science and Engineering Center Program Grant DMR
02-13805 and the National Institutes of Health Grant R01 HL069957.
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recently, it was believed that Ang-1 had solely a stabilizing role
via activation of the tyrosine kinase receptor Tie-2 (46), whereas
Ang-2, the antagonist to Ang-1, was believed to have more of a
facilitative role (47). For example, expression of Ang-2 was
identified primarily at sites of active vessel remodeling (47–50).
However, recent studies demonstrate that there may exist a
contextual role to the functions of Ang-2, because it serves in
some instances to inhibit vascular leakage (51), whereas in other
situations, it may function as a proinflammatory cytokine (52).
Increased secretion of Ang-2 in response to biochemical stimulants has also been documented, supporting the suggestion (53)
that Ang-2 function is more complex than initially identified
(54). Although VEGF-A and angiopoietins have distinct roles in
vascular development, they also have complementary and coordinated roles. VEGF-A has been shown to modulate migration
(32) and in vitro capillary formation (55) of ECs, and Ang-2, at
levels secreted in response to cyclic strain signals, appears to
have similar effects on ECs. Although the molecular mechanisms
linking cyclic strain to Ang-2 expression are not clear, they likely
involve the various intracellular signaling pathways previously
documented to mediate mechanical effects on ECs (16, 56) that
induce local differentiation and the formation of nascent blood
vessels.
Cyclic tensile strain was found in this study and by others (57)
to enhance the expression of PDGF-R␤ on HASMCs. A resulting enhanced responsiveness to PDGF signaling via the tyrosine
kinase pathway (activated by ligand binding to PDGF-R) is likely
involved in the HASMC migration in response to cyclic strain.
Cyclic strain has been previously demonstrated to modulate
ERK1/2 (58, 59), PI3K (60), p21 (19, 61), tyrosine kinase (59,
62), and RhoA signaling. Cyclic strain also likely enhances the
expression of other receptors as well, all which may cooperatively
regulate the HASMC responses to cyclic strain, including direct
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