Matrix-bound VEGF mimetic peptides: Design and endothelial-cell activation in collagen scaffolds

Article abstract of DOI:10.1002/adfm.201101163

Long-term survival and success of artificial tissue constructs depend greatly on vascularization. Endothelial-cell (EC) differentiation and vasculature formation are dependent on spatiotemporal cues in the extracellular matrix that dynamically interact with cells; a process that is difficult to reproduce in artificial systems. Here, a novel bifunctional peptide is presented that mimics matrix-bound vascular endothelial growth factor (VEGF) which can be used to encode spatially controlled angiogenic signals in collagen scaffolds. The peptide is comprised of a collagen mimetic domain that was previously reported to bind to type I collagen by a unique hybridization mechanism, and a VEGF-mimetic domain with pro-angiogenic activity. Circular dichroism and collagen-binding studies confirm the triple-helical structure and the collagen binding affinity of the collagen-mimetic domain, and EC-culture studies demonstrate the peptide's ability to induce endothelial cell morphogenesis and network formation as a matrix-bound factor in 2D and 3D collagen scaffolds. Spatial modification of collagen substrates is also shown with this peptide, which allows localized EC activation and network formation. These results demonstrate that the peptide can be used to present spatially directed angiogenic cues in collagen scaffolds, which may be useful for engineering organized microvasculature. Spatial modification of a 2D collagen substrate with a matrix-bound vascular endothelial growth factor (VEGF) mimetic peptide, QKCMP (CMP = collagen mimetic peptide), allows localized endothelial cell activation and network formation in a collagen matrix. Characterization and cell studies show that the peptide can bind to natural collagen and signal to endothelial cells in 2D and 3D cell constructs. QKCMP may allow spatially directed angiogenesis for tissue-engineering applications. Copyright

Full text of DOI:10.1002/adfm.201101163

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Matrix-Bound VEGF Mimetic Peptides: Design and
Endothelial-Cell Activation in Collagen Scaffolds
Tania R. Chan, Patrick J. Stahl, and S. Michael Yu*
model scaffolds tested to date, only natural
scaffolds, collagen and fibrin in particular,
have shown the consistent ability to form
thin-walled, fluid-filled endothelial cell
tubes with a close resemblance to natural
capillaries.[7] Unlike synthetic scaffolds,
scaffolds derived from natural ECM com-
ponents possess unique mechanochemical
signaling capabilities that regulate vascular
lumen formation and support long-term
vessel stability.[8,9]
The ECM is critical to vascular biology
as a scaffold for EC attachment and for
supporting chemotactic migration of
endothelial cells. As the primary protein
component of the ECM, collagen has
been identified as a regulator of angiogen-
esis. In concert with vascular endothelial
growth factor (VEGF), type I collagen has
been shown to facilitate multicellular reor-
ganization, activate actin polymerization,
and induce stress fiber formation in ECs,
all of which are necessary for capillary
morphogenesis.[10]
Long-term survival and success of artificial tissue constructs depend greatly
on vascularization. Endothelial-cell (EC) differentiation and vasculature
formation are dependent on spatiotemporal cues in the extracellular matrix
that dynamically interact with cells; a process that is difficult to reproduce
in artificial systems. Here, a novel bifunctional peptide is presented that
mimics matrix-bound vascular endothelial growth factor (VEGF) which can be
used to encode spatially controlled angiogenic signals in collagen scaffolds.
The peptide is comprised of a collagen mimetic domain that was previously
reported to bind to type I collagen by a unique hybridization mechanism, and
a VEGF-mimetic domain with pro-angiogenic activity. Circular dichroism and
collagen-binding studies confirm the triple-helical structure and the collagen
binding affinity of the collagen-mimetic domain, and EC-culture studies
demonstrate the peptide’s ability to induce endothelial cell morphogenesis
and network formation as a matrix-bound factor in 2D and 3D collagen
scaffolds. Spatial modification of collagen substrates is also shown with this
peptide, which allows localized EC activation and network formation. These
results demonstrate that the peptide can be used to present spatially directed
angiogenic cues in collagen scaffolds, which may be useful for engineering
organized microvasculature.
Despite the active role that the struc-
tural components of the ECM play in ang-
1. Introduction
iogenesis, development of new blood vessels is chiefly governed
by VEGF, which exists in three major isoforms—VEGF121
Angiogenesis and vascularization are vital for successful tissue
engineering, as newly engineered tissues require blood vessels
to deliver nutrients essential for long-term survival. However,
engineered tissues often lack the proper capability to form
functional vasculature,[1,2] because angiogenesis is a complex
biological process that requires a cascade of growth-factor sig-
naling and extracellular matrix (ECM) degradation to promote
proliferation, organization, and differentiation of endothelial
cells (EC). Although researchers have conjugated angiogenic
factors to tissue-engineering scaffolds to enhance angiogen-
esis,[3–6] most of these studies have been conducted using
simple scaffolds, such as synthetic polymers, that allow easy
chemical modification without concerns for nonspecific conju-
gation and scaffold deterioration. However, among numerous
,
VEGF165, and VEGF189. These three isoforms have different bio-
availability dictated by differing binding affinities to the ECM.[11]
The difference in binding affinity among the three VEGF iso-
forms is due to the presence or absence of a heparin-binding
domain, a sequence of fifteen basic residues encoded by exons
6 and 7 of the VEGF gene. VEGF189, a matrix-bound factor with
the highest ECM affinity of the three isoforms, contains the
heparin-binding domain as well as additional basic residues for
high heparin sulfate binding affinity. VEGF165 contains only the
heparin-binding domain and binds to the ECM with moderate
affinity. Finally, VEGF121 contains no heparin-binding domain
and exists solely as a soluble factor in the ECM. Together, the
three VEGF isoforms are believed to create a spatial and tem-
poral gradient that guides angiogenesis.
There have been multiple attempts to immobilize VEGF on
surfaces and scaffolds through the use of covalent conjuga-
tion chemistry (e.g., carbodiimide reaction),[12,13] or the use of
a crosslinker and binding tags.[5,14,15] However, these chemical
reactions are nonspecific and often require organic solvents
that can compromise VEGF activity and/or physicochem-
ical integrity of the scaffolds.[16,17] Moreover, these previous
T. R. Chan, P. J. Stahl, Prof. S. M. Yu
Department of Materials Science and Engineering
Institute for NanoBioTechnology
The Johns Hopkins University
3400 N. Charles St, Baltimore, MD 21218, USA
E-mail: yu@jhu.edu
DOI: 10.1002/adfm.201101163
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T a b le 1 . Amino acid sequences and molecular weights of synthetic
peptides.
immobilization studies were primarily intended for sustained
delivery of VEGF, which otherwise degrades quickly in physi-
ological settings.[16] These methods also lack the capacity to spa-
tially control the immobilization process, which may be neces-
sary for directed angiogenesis.
Molecular Weight [g mol−1
]
Peptide
QK
Sequence
Aca)-KLTWQELYQLKYKGI
1952
2421
4526
Alternatively, other research groups have sought to emu-
late VEGF and cell-receptor interactions using peptides and
antibodies.[18–22] One of the most promising pro-angiogenic
compounds comes in the form of a short peptide based on the
structure of VEGF; this was developed by d’Andrea et al., and is
a 15 amino acid sequence, referred to as the QK peptide, that
mimics the α-helical receptor binding region of VEGF.[23] This
peptide promotes attachment and proliferation of ECs, exhibits
competitive binding to VEGF receptors, and most importantly,
induces EC activation and capillarylike formation in vitro.[23–25]
QK peptide is promising not only for its extensive pro-ang-
iogenic activity, but also for its short peptide sequence that can
be easily synthesized and conjugated to various peptide delivery
vehicles.
b)
CMP
(POG)9
QKCMP
Ac-KLTWQELYQLKYKGIGGG(POG)9
a)Ac: acetyl; b)O: 4-hydroxyproline (Hyp).
melting temperature as well as higher collagen binding affinity
than those with fewer repeat units.[30] The varying degrees of
collagen affinity could be used to mimic the varying heparin
binding affinity of natural VEGF isoforms.
Here, we present a novel bifunctional peptide, QKCMP, com-
prised of a VEGF-mimetic QK domain and a collagen binding
CMP domain that can be used to control EC morphogenesis
in collagen scaffolds for tissue-engineering applications. We
characterize the thermal behavior of QKCMP, demonstrate its
binding affinity to type I collagen, and evaluate its morphogenic
effects on endothelial cells.
Although QK peptide has shown some therapeutic promise
in vivo, it has mostly been used as a locally delivered soluble
factor.[26,27] There have been attempts to immobilize QK on
inorganic substrates[25] and self-assembling peptide scaffolds,[24]
and recently, QK has been conjugated to bioactive hydrogel
through poly(ethylene glycol) linkers[17] and to hydroxyapatite
through a binding peptide.[28] These two recent studies have
shown that the immobilized form of QK retains its biological
function and is able to elicit EC response. However, these sub-
strates lack the dynamic physicochemical signaling capabilities
of collagen, and are not optimal for supporting long-term func-
tional vessel growth in vitro. We hoped to create a QK peptide
that can bind to a collagen scaffold similar to VEGF189 binding
to heparin, and evaluate this peptide’s ability to act as a matrix-
bound VEGF-like signaling factor in natural collagen scaffolds
which are widely used for engineering microvasculatures.
To achieve collagen affinity, we employed a collagen-mimetic
peptide (CMP) sequence which has been reported to bind to
type I collagen with varying degrees of affinity.[29–32] CMPs are
a widely used model system in studying the triple-helical struc-
ture of natural collagen. CMP contains the Gly-X-Y sequence
commonly found in natural triple-helical collagen, with a
majority of the X and Y positions populated, respectively, by
hydroxyproline (Hyp) and proline (Pro). This unique sequence
allows CMPs to form triple-helical supermolecular structures
nearly identical to those in natural collagen. Unlike natural
collagen, however, these peptides form thermally reversible
triple helices. Due to their small size (<3 kDa), CMPs exist in a
single-stranded conformation when heated above their melting
temperature and reform triple-helical structures upon cooling.
We have previously demonstrated a method to bind and modify
natural collagen by addition of melted, single-stranded CMPs
to a preformed collagen matrix.[30–32] The binding took place
when single-stranded CMPs were allowed to fold by cooling in
the presence of type I collagen. Binding of CMPs to collagen
films and gels is driven by hybridization interactions believed
to be mediated by strand invasion and triple-helical associa-
tion. The melting temperature and collagen binding affinity of
CMPs can be tuned by varying the number of repeating Gly-X-
Y units. CMPs with more Gly-X-Y repeating units have a higher
2. Results and Discussion
2.1. Design and Characterization of QKCMP
As shown in Table 1, the target peptide designated as QKCMP
consists of the VEGF-mimetic QK domain and the collagen-
binding CMP domain. The QK domain was coupled to the
N-terminal side of the CMP with a three-glycine spacer. We
chose (ProHypGly)9 as the CMP domain because of its high
collagen binding affinity.[30,31] As control samples, the QK and
CMP domains were also produced separately. The peptides were
prepared manually by means of F-moc mediated solid-phase
peptide synthesis and purified using high-performance liquid
chromatography. We used circular dichroism (CD) to investi-
gate the structural characteristics of the QK, CMP, and QKCMP
peptides (Figure 1a). As expected, CMP and QK displayed the
characteristic CD spectra of triple[31,33] and alpha helices,[23]
respectively, with CMP showing a maximum ellipticity at 225 nm
and QK showing double minima at around 208 and 222 nm. The
QKCMP signal corresponded to a sum of the CMP and QK CD
signals, which suggests that the structures of the two domains
were intact after the conjugation. Even under acidic pH condi-
tions, QKCMP maintained the same CD spectra (Figure 1b). As
previously reported, CMP’s binding affinity to natural collagen
is primarily driven by its propensity to form a triple helix.[30] The
ability of QKCMP to retain its structure in acidic conditions sug-
gests that the peptide can be used to bind to collagen in acidic
conditions without concerns about denaturation.
2.2. Thermal Folding Behavior of QKCMP
Having identified QKCMP’s triple-helical structure, we investi-
gated the thermal melting and folding behavior of the peptide
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binding of thermally quenched CMPs (CMPs
first heated to temperature above its Tm
and quickly cooled to room temperature) to
natural collagen by taking advantage of the
slow refolding rate of CMPs, which provided
a time window for single-stranded CMP
to hybridize with collagen before folding
into homotrimeric CMP triple helices.[36]
To study the folding rate of QKCMP, we
heated QKCMP to 80 °C followed by a quick
drop in temperature to 37 °C, and moni-
tored the changes in CD ellipticity at 37 °C
(Figure 1d). As expected, QKCMP exhibited
a slow refolding rate with t1/2 close to 1 h,
which indicates that the peptide’s folding
process is similar to other CMPs, and that
quenched single strands of QKCMP can
hybridize with natural collagen.
2.3. Immobilization and Sustained Release of
QKCMP in Collagen Gels and Coatings
In previous experiments,[30,31,36] collagens
were modified with CMPs by adding hot or
quenched CMPs to preformed collagen films
and gels. Recently, we discovered high CMP
immobilization when CMPs were added to
the collagen solution before film and gel for-
mation (Supporting Information, Figure S1).
Figure 1. a) Full CD spectra of CMP, QK, and QKCMP in phosphate-buffered saline (PBS).
b) Full CD spectra of QKCMP at different pH values. c) CD melting curves of CMP, QK, and
QKCMP. d) Triple-helix refolding curve of QKCMP at 37 °C.
Rather than allowing only surface binding to take place, this
new method exposes CMPs to the bulk collagens and thereby
maximizes the collagen loading level. The films and gels con-
taining collagen fibers were formed by neutralization of acidic
collagen solution followed by incubation at 37 °C. Figure 2a
shows the SEM and TEM (inset) images of type I collagen fibers
prepared from a collagen solution (1.8 mg mL−1 ) mixed with
quenched QKCMP (25 μM). The morphology of collagen fibers
is nearly identically to that prepared from pure collagen solu-
tion (Figure 2b), which suggests that the presence of QKCMP
did not interfere with collagen-fiber formation. Films and gels
fabricated from QKCMP and collagen solution have a physical
appearance and handling characteristics similar to those made
of pure collagen. It seems that CMP addition does not interfere
with the collagen gelation process because the gel starts to form
within a few minutes, while CMP folding and collagen hybridi-
zation may take over an hour. It is also possible that the fiber
formation, which is driven by strong secondary forces such as
charge–charge interactions and hydrophobic interactions, can
dominate the relatively weak CMP–collagen hybridization inter-
action and that the CMP–collagen hybridization becomes fully
active only after collagen fibers have already developed.
by monitoring QKCMP’s triple-helical CD signal as a function
of temperature. Figure 1c shows the CD melting curves for
CMP, QK, and QKCMP, which track their respective ellipticity
at 225 nm as a function of temperature. The melting curve
of CMP exhibited a sigmoidal shape indicative of a two-state
melting transition, as reported earlier.[30,31] In contrast, the
heating of the QK peptide did not produce a sigmoidal curve,
and no melting transition was observed. This result is in agree-
ment with the previous reports that QK retains its alpha-helical
form even at high temperatures.[34] The peptide of interest,
QKCMP, also exhibited a triple-helical melting transition as
demonstrated by the sigmoidal melting curve in Figure 1c. By
determining the minimum of the first derivatives of the sig-
moidal curves,[35] the melting temperatures (Tm) of CMP and
QKCMP were identified as 69 °C and 67.5 °C, respectively. The
slightly lower melting temperature of QKCMP is likely due to
the coulombic repulsion between the charged QK domains.
The well-defined triple-helical melting transition suggests that
QKCMP could bind to type I collagen by the triple-helical pro-
pensity-driven hybridization mechanism previously shown for
CMP and other CMP derivatives.[30–32,36]
After establishing its melting behavior, we focused on the
refolding kinetics of QKCMP. Compared to other secondary
protein structures, the collagen triple helix is known to have a
slower folding rate, typically on the order of minutes to hours.[33]
This is because of the slow cis–trans isomerization speed of
Pro and Hyp, and a multistep folding process involving chain
association and registration, followed by triple-helix nuclea-
tion and propagation.[37–39] Previously, we have demonstrated
QKCMP (labeled with carboxyfluorescein at the N terminus)
showed collagen-binding characteristics and sustained-release
behavior similar to those previously reported for CMP derivatives
(Figure 2c and d).[30,36] After collagen coating or gel formation,
unbound peptides were removed from the substrates by washing
with excess buffer solution. The amount of peptide bound to
the collagen substrates was determined using fluorescence
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(CF), the level of initial binding after wash was approximately
one third of that of the QKCMP. This background binding is
likely due to trapping effects within the pores of the collagen
gel and nonspecific charge–charge interactions with type I col-
lagen. Over the next four days of 37 °C incubation, we observed
a gradual release of QKCMP from the collagen gel, with 30%
(1.5 nmol) remaining on day 2 and 20% (1 nmol) remaining on
day 4 (Figure 2c). We did not see significant changes in peptide-
release behavior on subsequent days. In contrast, the amounts
of CF-labeled QK and CF in the collagen gel quickly dropped
to below 10% (<0.5 nmol) on day 2. In the case of type I col-
lagen coating, 1.8 nmol of CF-labeled QKCMP, which corre-
sponds to a density of 5.53 0.68 nmol cm−2 , remained bound
to the substrate compared to 0.6 nmol of CF and CF-tagged QK
(Figure 2d). In 24 h of observation, we saw very little subsequent
release of the peptide from the collagen coating.
The binding and release study indicates that the CMP domain
of QKCMP retains collagen-binding capability and can turn a
soluble QK molecule into a collagen-bound factor similar to the
action of the heparin-binding domain of VEGF. Although there
have been previous attempts to immobilize VEGF onto 2D sur-
faces, those studies only achieved low ligand density ranging
from 3 fmol cm−2 to 6 pmol cm−2 .[15,17,36,37,40,41] The bulkiness
of the VEGF protein, as well as the usage of intermediary linker
molecules in these studies, likely contributes to the low binding
density. In comparison, immobilization of the small bioactive
QK peptide onto 2D surfaces using covalently attached linkers
produced significantly higher surface density, ranging from
3 pmol cm−2 to 2.6 nmol cm−2 .[17,28] Our binding studies dem-
onstrated that QKCMP can also bind to 2D collagen substrates
at a even higher density (5.53 0.68 nmol cm−2 ), and we expect
to see pronounced morphological changes in EC exposed to
QKCMP immobilized on collagen scaffolds.
2.4. Endothelial Cell Morphogenesis in 2D and 3D
Collagen Scaffolds
Next, we tested the ability of QKCMP to signal to ECs to induce
tubular morphogenesis in 2D and 3D environments. In nature,
angiogenesis relies on a signaling cascade comprised of both
soluble and insoluble growth factors. We employed QKCMP to
mimic the matrix-bound VEGF isoforms by creating a peptide
that has an EC signaling domain and an ECM binding domain.
We performed extensive morphogenesis studies using type
I collagen coatings containing QKCMP. The collagen coatings
were prepared by the same method as used for the release study
and the human umbilical-vein endothelial cells (HUVECs)
were plated at 15,000 cells per well in a 96-well plate. As seen
in Figure 3a, HUVECs plated on collagen coatings modified
with QKCMP (25 and 100 μM of QKCMP in 0.1 mg mL−1 col-
lagen coating solution) exhibited network morphology, while
comparatively, HUVECs plated on plain collagen coating or col-
lagen coating with QK added in the same manner (100 μM QK
in 0.1 mg mL−1 collagen coating solution) displayed only cobble-
stone morphology. This observation indicates that QKCMP,
when immobilized to the collagen substrate through the CMP
domain, is able to elicit a unique EC response. On the coating
with the highest QKCMP concentration (100 μM QKCMP in
Figure 2. SEM and TEM (insets) images of a) QKCMP-modified type I
collagen gel and b) pure type I collagen gel. Cumulative release profiles
of CF-QKCMP, CF-QK, and CF from c) type I collagen gels and d) type
I collagen coatings. Initially, 5 and 10 nmol of each sample were added
to each collagen gel and collagen coating, respectively. Day 0 and hour
0 represent the amount of compounds remaining in the collagen matrix
after removal of unbound peptide by washing with 4°C blank buffer. Error
bars represent SD.
spectrometry. In the case of the collagen gel (Figure 2c) about
3 nmol were bound to the collagen gel after the initial wash;
this corresponds to 60% of the QKCMPs initially applied and
correlates to roughly one QKCMP molecule for every one hun-
dred collagen molecules. Considering that the diameter of a col-
lagen fiber is approximately 100 to 200 times that of a collagen
molecule, we believe that the surfaces of collagen fibers are
densely decorated with QKCMP. For QK and carboxyfluorescein
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Figure 3. a) Phase-contrast micrographs of HUVECs plated on type I collagen coatings modified with varying concentrations of QKCMP and QK.
b) Fluorescence micrographs of HUVECs stained with anti-PECAM-1 monoclonal antibodies (red), phalloidin (stains actin in green), and DAPI (stains
nucleus in blue) plated on QKCMP modified collagen (left panel) and plain collagen coatings (right panel). Filopodia extensions are marked by arrows.
Bar represents 100 μm.
0.1 mg mL−1 collagen coating solution), HUVECs began to show
networklike developments in as little as 3 h. As the concentra-
tion of QKCMP decreased, network morphology did not develop
until later; 6 h in the case of intermediate QKCMP concentration
(25 μM QKCMP in 0.1 mg mL−1 collagen coating solution) and
50 h in the case of low QKCMP concentration (data not shown).
HUVECs plated on plain collagen coating or on collagen coating
with QK (100 μM QK in 0.1 mg mL−1 collagen coating solution)
did not show any network morphological development during
the 50 h time-frame. Although high QKCMP concentrations
were able to quickly induce morphological changes, HUVECs
stimulated under those conditions continued to migrate even
after the initial network formation, and formed inactive isolated
cell clusters after more than 24 h incubation.
of high concentrations of platelet-endothelial cell-adhesion
molecules (PECAM) dispersed throughout the cell surfaces
as evidenced by immunofluorescence microscopy (Figure 3b).
PECAM is present at cell junctions of quiescent blood vessels,
inhibiting EC migration and promoting stable EC–EC junc-
tions.[42–44] However, in angiogenic ECs, PECAM is redistrib-
uted from cell junctions to cell surfaces to activate EC migra-
tion.[45–47] The diffuse presence of PECAM on the EC surface
is a strong indication that the cells’ angiogenic pathways have
been activated.
Furthermore, as shown in phalloidin/4’,6-diamidino-2-phe-
nylindole dihydrochloride (DAPI) staining, the network mor-
phology of HUVECs incubated on QKCMP-modified collagen
substrate occurred in concert with the development of filo-
podia extensions in multiple directions (arrows in Figure 3b).
Such filopodia extensions were not observed in the cells with
The network morphology of HUVECs incubated on QKCMP
modified collagen coating was accompanied by the presence
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Figure 5. Effects of QKCMP-modified type I collagen on a) the expression
and b) phosphorylation of ERK1/2 in HUVECs. Western blot experiments
show that neither QKCMP nor soluble VEGF (100 ng mL−1 ) affect the
expression levels of ERK (a), while increasing the phosphorylation level
of ERK (Student’s t test, p < 0.01). Error bars represent SD.
increasing EC proliferation and promoting EC migration.[23,49–51]
To verify that the EC migration and morphological changes
that we observed are due to ERK1/2 activation by QKCMP, we
examined the ERK1/2 phosphorylation levels of ECs that were
incubated for 1 h on the QKCMP-modified collagen substrates
(Figure 5). In comparison to cells grown on plain collagen sub-
strate (0.1 mg mL−1 collagen coating solution), cells seeded on
QKCMP-modified collagen (100 μM QKCMP in 0.1 mg mL−1
collagen coating solution) showed a marked increase in phos-
phorylation level (Figure 5b), while maintaining the same level
of ERK1/2 expression (Figure 5a). The phosphorylation level
shown by the cells on the QKCMP-modified surface was not
as high as that of the cells seeded on plain collagen supple-
mented with 100 ng mL−1 of soluble VEGF, which is similar to
findings by those who originally developed QK.[23] This result
indicates that the immobilized QKCMP retains the bioactivity
of the QK and is able to activate the canonical pathway in VEGF
signaling.
The network morphology and viability of HUVECs activated
by collagen-bound QKCMP can be prolonged by the addi-
tion of soluble VEGF. As seen in Figure 6a, when cell-culture
medium was supplemented with 500 ng mL−1 of soluble VEGF,
the networklike morphology of HUVECs seeded on QKCMP-
modified collagen coating (100 μM QKCMP in 0.1 mg mL−1
collagen coating solution) was maintained up to 24 h. This
result is in contrast to cells exposed to only QKCMP, which
form inactive cell clusters before 24 h. Furthermore, prolifera-
tion-rate assays (Figure 6b) indicated increased EC proliferation
in the presence of soluble VEGF. Although previous studies
involving soluble VEGF have shown that 100 ng mL−1 elicits
maximum cellular response,[16,52,53] this concentration was not
able to prolong the viability of HUVECs which were also acti-
vated by our collagen-bound QKCMP. This observation is most
likely due to changes in VEGF-receptor response in the pres-
ence of both soluble and matrix-bound factors, and more work
is currently in progress to elucidate this phenomenon.
Figure 4. Phase-contrast micrographs of human dermal neonatal fibrob-
lasts plated on plain collagen coating (0.1 mg mL−1 coating solution) and
QKCMP-modified collagen coating (100 μM of QKCMP in 0.1 mg mL−1
coating solution). Bar represents 100 μm.
cobble-stone morphology (plain collagen and QK-supplemented
collagen). VEGF has been known to direct filopodial behavior
where ECs extend thin actin processes to form stress fibers in
the direction of VEGF signals for vascular expansion.[42] Model
animals that only express the heparin-binding form of VEGF
have been shown to develop small caliber blood vessels with
excessive branching, which is preceded by the development of
excess filopodia in multiple directions and prolonged EC migra-
tion.[42,43,48] The presence of PECAM on cell surfaces, combined
with multidirectional filopodia development and continued cell
migration after initial network formation, strongly suggests
that QKCMP is giving matrix-bound VEGF-like signals to ECs
in collagen substrates.
To confirm that the enhanced HUVEC morphogenesis on
QKCMP-modified collagen coatings was induced by EC specific
stimulation, we conducted the same morphological studies with
fibroblasts. Fibroblasts (human dermal neonatal fibroblasts)
plated on both plain and QKCMP-modified collagen coatings
displayed identical fusiform morphology after 24 h (Figure 4).
The absence of morphological transformation on these fibrob-
lasts suggests that the QKCMP’s effect on HUVEC morphology
did not result from generic cell–substrate interactions (e.g.,
mechanical stiffness or charge interactions) but instead devel-
oped specifically from endothelial cell stimulation by the pep-
tide’s QK domain, possibly mediated by VEGF receptors.
Extracellular signal-regulated kinases 1 and 2 (ERK1/2) play
a crucial role in the VEGF-dependent activation of ECs. Phos-
phorylation of ERK1/2 is known to elicit EC angiogenesis by
The angiogenic activity of QKCMP in the presence of sol-
uble VEGF was also observed in the 3D cell construct. We
encapsulated EC spheroids[54,55] in collagen gels that con-
tained QKCMP (25 μM) and observed tubes sprouting from
EC spheroids. According to the QKCMP immobilization study
(Figure 2), we expected approximately 3 nmol of immobilized
QKCMP initially and 1 nmol of QKCMP released from the
collagen gel during the EC sprouting experiment. Since our
2D culture study indicated that the soluble form of QKCMP
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gels induced EC sprouting, possibly in response to soluble
VEGF supplemented to the culture media, spheroids encap-
sulated in QKCMP-containing collagen gels exhibited greater
sprouting with more elongated tube morphology, as compared
to fewer and flattened sprouting morphology in the pure col-
lagen gels. This result was confirmed by examining the total
number and total length of the sprouts per spheroid (Figure 7c
and d). The results suggest that the collagen-bound QKCMP
and soluble VEGF work synergistically in a manner similar to
insoluble and soluble VEGF in native tissue to stimulate EC
sprouting and tube elongation, which are early indications of
angiogenesis.
Previous research demonstrated that matrix-bound iso-
forms of VEGF promote cell migration and new vessel forma-
tion while soluble isoforms of VEGF induce EC proliferation
and vessel maturation.[42,56] Model animals expressing only the
matrix-bound form of VEGF suffer from hypervascularization.
Such models develop abnormally thin blood vessels character-
ized by excessive branching at the expense of lumen growth
and are more likely to die in utero.[42,43,56] The combinatorial
expression of both matrix-binding and soluble VEGF creates a
spatial gradient that guides EC migration, vascular formation,
and branching development to form fully functional vascular
networks. Our experiments showed the ability of collagen-
bound QKCMP to activate EC morphogenesis, causing ectopic
filopodia development and excessive cell migration that mirrors
the beginning stages of excessive vessel branching in animals
that express only matrix-bound VEGF. The sustained network
development and sprouting of HUVECs grown in two- and
three-dimensional QKCMP-modified collagen substrates sup-
plemented with soluble VEGF suggest that QKCMP could be
used as a substitute for matrix-bound VEGF to engineer micro-
vasculature in collagen scaffolds.
Figure 6. Phase-contrast micrographs of HUVECs plated on QKCMP-
modified type I collagen a) with and without 500 ng mL−1 soluble VEGF.
b) WST-1 proliferation assay shows increased cell proliferation in cells
plated on QKCMP-modified collagen with 100 ng mL−1 of soluble VEGF
(Student’s t test, p < 0.05). Error bars represent SD. Bar represents
100 μm.
Figure 7. Phase-contrast microscopy images of 3D endothelial cell
spheroids (1 day culture) encapsulated in type I collagen gel supple-
mented with a) 2.5 ng mL−1 of soluble VEGF or b) 5 nmol QKCMP and
2.5 ng mL−1 soluble VEGF. Quantification of sprouting from spheroids
by c) the number of sprouts per sphere and d) total sprout length per
sphere. (Student’s t test: ∗ p < 0.01, ∗∗ p < 0.05) Error bars represent
SD. Bar represents 100 μm.
had no morphogenic effects on ECs, we expected similarly
that the QKCMP released during the experiment would have
little effect on ECs in 3D collagen gel. In Figure 7a and b, we
show comparative photos of EC spheroids undergoing tubular
morphogenesis in pure collagen and collagen with matrix-
bound QKCMP, on addition of VEGF. Although both collagen
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two halves of the well (Figure 8a). The cells were homogenously
dispersed within the well with a slightly rounded morphology,
as the cells were just beginning to attach to the collagen sub-
strate. The difference in morphology became apparent after
3 h incubation, where the cells on the plain collagen coating
had a cobble-stone morphology while the cells on the QKCMP-
modified collagen coating appeared elongated and were starting
to make cell–cell connections. The morphological differences
became more apparent after 6 h, as cells on the plain collagen
retained the cobble-stone morphology, while extensive network
formation was seen on the QKCMP-modified half of the well.
In Figure 8b, phalloidin/DAPI staining shows cobble-stone
morphology on the HUVECs plated on the plain collagen half
of the well. On the QKCMP-modified half, the cells show a
completely different morphology—they appear more active, as
filopodia protrusions are clearly visible from a majority of the
cells, indicative of cell movement and network formation. The
morphological differences between the two halves of the well
were maintained up to 24 h.
The morphologies of the HUVECs plated on the plain col-
lagen and QKCMP-modified surfaces are comparable to the
results discussed in the previous sections. The patterning
experiment, however, highlights the novelty of our system: for
the first time, we demonstrated that the EC morphology on col-
lagen coating within a single tissue-culture well can be spatially
controlled through the use of QKCMP peptides that mimic the
ECM binding and angiogenic activities of matrix-bound VEGF.
We were able to induce local EC network formation which
holds great promise for patterning tissue scaffold for directed
angiogenesis.
3. Conclusions
Recognition of the critical roles that the various VEGF isoforms
play in the development of vasculature prompted us to develop
a new peptide that can mimic the matrix-bound VEGF iso-
form (VEGF189), which is known to have high binding affinity
for ECM. We successfully designed and synthesized QKCMP,
a bifunctional peptide that contains an EC-activating domain
(QK) and a collagen-binding domain (CMP). Our studies
showed that this peptide folds into a triple-helical structure and
can bind to type I collagen when allowed to fold in the pres-
ence of collagen, similar to previous CMP derivatives, as previ-
ously reported.[30–32,36] Over 1 nmol QKCMP was immobilized
on type I collagen coatings covering the 96-well culture plate.
ECs plated on the QKCMP-immobilized collagen coatings
exhibited pro-angiogenic behaviors as evidenced by diffusive
presence of PECAM, multiple filopodia protrusions, and cell
migration which resulted in the development of cellular net-
works. EC sprouting studies of EC spheroids in 3D collagen
gel showed elevated tubulogenic behavior for QKCMP-modified
collagen gel. We also demonstrated that such cell morphogen-
esis can be induced in predefined areas within wider collagen-
based cell culture constructs by spatially controlling the areas of
QKCMP immobilization. The ability to encode spatially defined
morphogenic signals in natural scaffolds is expected to pro-
vide new pathways for engineering complex tissues for regen-
erative medicine. While others have immobilized VEGF and
Figure 8. Morphology of HUVECs plated on cell culture well half-coated
with plain type I collagen and half coated with QKCMP-modified type I col-
lagen as visualized by a) phase-contrast microscopy and b) fluorescence
microscopy after phalloidin and DAPI staining. Bar represents 100 μm.
2.5. Local Activation of ECs on Collagen Substrates
One advantage of our QKCMP system in activating ECs
is the ability to locally control the area of peptide binding,
which thereby allows spatial control of EC migration and net-
work morphology development. We coated half of a well (48-
well plate) with plain collagen (0.1 mg mL−1 collagen coating
solution) and the other half with QKCMP-modified collagen
(100 μM QKCMP in 0.1 mg mL−1 collagen coating solution).
After HUVEC seeding (55,000 cells per well), the well was sup-
plemented with 500 ng mL−1 of soluble VEGF. After 30 mins,
there was essentially no morphological difference between the
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VEGF-mimetic peptides to matrices,[16,17] the work presented
herein, along with the work by Lee and co-workers,[28] is one
of the first attempts in designing and studying peptides that
directly mimics not only the angiogenic activity but also the
ECM binding affinity of matrix-bound VEGF with the intent
to enhance or spatially direct microvasculature formation. In
the long run, the use of this matrix-bound VEGF mimetic may
allow production of organized vasculatures that are more akin
to natural tissues.
one of the three buffer solutions: 1 × PBS (pH 7.5), phosphate buffer
(pH 5.3), and acetic acid (pH 3.3). The concentrations of the samples
were determined by UV-Vis spectrophotometry at 275 or 493 nm. CD
spectra were measured on a JASCO 710 spectrometer with JASCO
PTC-348 WI temperature controller (Easton, MD). For thermal melting
curves, the molar ellipticity at 225 nm was monitored between 5 °C and
90 °C with a heating rate of 1 °C min−1
.
Determination of Refolding Yields of QKCMP: To measure the refolding
rate, QKCMP (100 μM) preincubated at 80 °C for 30 mins was quenched
to 37 °C in an ice bath and the change in molar ellipticity at 225 nm was
monitored for 3 h at 37 °C. The refolding yield was determined from the
following equation:
(
2 )
C at 225 nm
2A
−
4. Experimental Section
Refolding Yield
=
2B − 2C
(
)
at 225 nm
Materials: All amino acids and peptide synthesis reagents including
N-methylpyrrolidone (NMP), 2-(1H-benzo-triazole-1-yl)-1,1,3,3-tetra-
methyluronium hexafluorophosphate (HBTU), and trifluoroacetic acid
(TFA) were purchased from Advanced ChemTech (Louisville, KY). Rink-
type TentaGel R Ram resin was purchased from Peptides International
(Louisville,KY).Carboxyfluorescein(CF),piperidine,andtriisopropylsilane
(TIS) were purchased from Sigma-Aldrich (St. Louis, MO). Acid-soluble
rat-tail type I collagen was obtained from BD Bioscience (San Jose, CA).
All electron microscopy fixatives and staining agents were obtained from
Electron Microscopy Sciences (Hatfield, PA). HUVECs and endothelial
cell media were obtained from Lonza (Walkersville, MD), and VEGF
(VEGF recombinant human) was obtained from Invitrogen (Carlsbad,
CA). Fibroblasts (human dermal, neonatal) were purchased from
American Type Culture Collection (Manassas, VA) and fibroblast media
were obtained from Invitrogen. WST-1 proliferation assay was obtained
from Roche Applied Science (Indianapolis, IN). For Western blots, all
primary and secondary antibodies were obtained from Cell Signaling
Technologies (Beverly, MA). For immunohistochemical staining, 10%
neutral buffered formalin was obtained from Sigma-Aldrich, tris-buffered
saline (TBS) was purchased from Bio-Rad Laboratories (Hercules, CA),
and monoclonal mouse anti-human CD31/PECAM-1 primary antibody
and Cy3 conjugated anti-mouse secondary antibody were purchased
from BD Bioscience and Jackson ImmunoResearch Laboratories Inc
(West Grove, PA). Alexa-Fluor 488 phalloidin and 4,6-diamidino-2-
phenylindole (DAPI) were purchased from Invitrogen and Roche Applied
Science, respectively.
Peptide Synthesis: All peptides were synthesized by conventional solid-
phase peptide-synthesis methods on TentaGel R Ram resin in NMP using
4 molar excess of Fmoc-protected amino acids activated with HBTU.
Fmoc protection groups were removed with 20% piperidine in NMP. The
efficacy of the coupling and deprotection reactions was monitored with
ninhydrin and chloranil tests. For nonfluorescently labeled peptides, the
N-terminus of the peptide was capped with an acetyl group by treatment
with acetic anhydride. After completing the synthesis of the target
sequence, the peptide and all side-chain protection groups were cleaved
by treating the resin with a cleavage cocktail (95% TFA, 2.5% deionized
water, and 2.5% TIS) for 3 h. The crude peptides were precipitated by
addition to cold diethyl ether and dried in a vacuum.
where θA = Molar ellipticity at 225 nm of quenched sample at a given
time (helical content of quenched sample during refolding), θB = molar
ellipticity at 225 nm of sample after overnight incubation at 4 °C (helical
content of fully folded sample), and θC = molar ellipticity at 225 nm of
sample after 3 h incubation at 80 °C (helical content of fully unfolded
sample).
Immobilization of QKCMP in Collagen Gels and Coatings: To immobilize
QKCMP to collagen, CF-labeled QKCMP solution (200 μL, 25 μM)
was heated to 80 °C for at least 30 mins to ensure melting. The hot
solution was quenched to 37 °C in an ice-water bath and immediately
added to acid-soluble rat-tail type I collagen (100 μL). The mixture
solution was neutralized with dilute sodium hydroxide (100 μL), added
to a 48-well cell-culture plate, and incubated at 37 °C for 3 h to allow
gel formation and peptide binding. The final volume of the gel was
400 μL with 1 mg mL−1 collagen concentration. The collagen gel was
then washed ten times, using 500 μL 4 °C PBS, to remove any unbound
peptides. After the wash, each well was reloaded with 500 μL PBS and
incubated at 37 °C for the release experiment. The PBS solution was
replaced every two days.
To determine the amount of peptide bound to the collagen gels, the
fluorescence intensity of the gels (after melting) was measured with
a Gemini EM Fluorescence Microplate Reader (Molecular Devices,
Sunnyvale, CA). For each time point in the binding release study, excess
PBS was first removed from the collagen gel. The remaining collagen
gels were melted at 80 °C for 45 mins. The solutions were then cooled
to 4 °C and their fluorescence intensities were measured at 533 nm
emission and 489 nm excitation wavelengths. The fluorescence readings
were then converted to a concentration of CF-labeled peptides by using
a standard curve generated for each type of peptide studied.
To measure QKCMP binding to the collagen coating, a 1 mM stock
solution of CF-labeled peptide solution was created in PBS. The solution
was heated at 80 °C for at least 30 mins to enable triple-helical melting.
Separately, a collagen solution was created by mixing rat-tail type I collagen
with PBS to give a final collagen concentration of 0.1 mg mL−1 . The heated
CF-labeled peptide solution was then added directly to the collagen coating
solution, to create a final peptide concentration of 100 μM. No quenching
was required since the heated peptides were quickly cooled during the
mixing step due to the large volume of collagen coating solution (10 to
40 fold dilution). The mixture solution (100 μL) was added to the wells
of a 96-well plate and incubated for 1 h. After the incubation, to ensure
that the unbound peptides were removed from the collagen-coated well,
all excess solutions were removed from the cell-culture well and saved for
fluorescence readings. The wells were then replenished with 100 μL fresh
PBS. Subsequently, the culture plate was incubated at 37 °C and each
well was replaced with 100 μL fresh PBS at each noted time-point for the
peptide-release study. The PBS buffer removed during the study was saved
for fluorescence readings. The amount of CF-labeled peptide remaining
in the collagen coating was determined from the fluorescence intensities
of the released peptides in the PBS solutions, which were converted to a
concentration using a custom calibration curve.
The crude peptides were purified using semipreparative reverse-phase
high-performance liquid chromatography (HPLC) on a Varian Polaris
210 series liquid chromatograph (Agilent Technologies, Santa Clara,
CA) with UV detection at 275 nm (for general peptides) or 493 nm (for
CF-containing peptides), using a Vydac C-4 reverse-phased column from
Grace Davison Discovery Sciences (Deerfield, IL). The HPLC was run at a
flow rate of 4 mL min−1 using a solvent gradient comprised of deionized
water (0.1% TFA) and methanol (0.1% TFA). The elutions corresponding
to the target peak in the chromatogram were combined and lyophilized.
The purified peptides were analyzed by matrix-assisted laser desorption/
ionization time-of-flight (MALDI-ToF) mass spectrometry (Voyager
DE-STR, Applied Biosystems, Foster City, CA.) The mass spectra of all
peptides are shown in Figure S2.
Circular Dichroism and Thermal Melting Experiments: Prior to CD
measurements, peptides (100 μM) were incubated at 4 °C overnight in
Electron Microscopy: QKCMP-modified collagen gel (400 μL with
1.8 mg mL−1 collagen concentration) was created as described above.
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Plain collagen gel was created by the same method, but PBS was used
instead of the QKCMP solution. The gels were first fixed for 1 h with
100 mM sodium cacodylate solution containing 3% paraformaldehyde,
1.5% glutaraldehyde, and 2.5% sucrose, and stained with 1% osmium
tetroxide solution for 30 min. For SEM, the samples were dehydrated
with ethanol and dried with a supercritical point dryer (Tousimis 795,
Rockville, MD). The samples were then sputter-coated with a 4-nm thick
platinum layer (Anatach Sputter Coater, Union City, CA) and observed
under high vacuum in FEI Quanta ESEM 200 (Hillsboro, OR). For TEM,
the samples were additionally stained with Kellenberger’s uranyl acetate
overnight before ethanol dehydration. The dehydrated samples were
then embedded in Embed 812 resin and sectioned into 80–100 nm thick
slices. The sample slices were placed on a TEM grid and observed with
FEI Tecnai 12 (Hillsboro, OR).
Cell Culture: HUVECs were cultured in a complete endothelial-cell
growth medium (EGM-2) and human dermal neonatal fibroblasts were
cultured in 1:1 Dulbecco’s Modified Eagle Medium/Ham’s F-12 Nutrient
Mixture (DMEM/F-12 with GlutaMAX, Invitrogen) supplemented
with 10% fetal bovine serum (FBS). Both cell types were grown at
37 °C with a fully humidified atmosphere and 5% CO2. The growth
media were changed every other day. The cells were harvested with
trypsin-ethylenediaminetetraacetic acid (EDTA) treatment at 70%–80%
confluency, and only passage 2-7 cells were used in the 2D and 3D
tubulogenesis experiments. All phase-contrast images were taken with
an EVOS phase-contrast microscope (Advanced Microscopy Group,
Botwell, WA).
electrophoresis. The separated proteins were then transferred onto a
nitrocellulose membrane using the iBlot Dry Blotting System (Invitrogen).
The membrane was blocked with 3% milk for 1 hr at room temperature,
and then stained overnight at 4 °C with specific antibodies for total ERK1/2,
and Phospho-p44/42 ERK1/2. Afterwards, the membranes were stained
with anti-rabbit horseradish peroxidase conjugated secondary antibody for
1 h at room temperature. Amersham ECL detection reagent (GE Healthcare
Life Sciences, UK) was used to reveal the bands on films, and the bands
were quantified using Amersham ImageQuant TL (GE Healthcare Life
Science). Data and analysis were taken from three independent trials.
Endothelial-Cell Sprouting in 3D Collagen Gels: Endothelial-cell
spheroids for 3D tubulogenesis study were constructed as previously
described.[46,47] Briefly, methylcellulose was dissolved in 30 mL of
EGM-2 at a concentration of 24 mg mL−1 . HUVECS were suspended in
the methycellulose/EGM-2 solution and incubated overnight in round-
bottomed 96-well plates (500 cells well−1 ) to allow spheroid formation.
Spheroids were harvested with a multichannel pipette and dispersed in
neutralized type I collagen solution (1.8 mg mL−1 ) or QKCMP-modified
collagen solutions (25 μM QKCMP in 1.8 mg mL−1 collagen) prepared
as previously described. The spheroid–collagen solution was delivered
to the wells of 48-well plates (200 μL) and incubated at 37 °C for gel
formation and spheroid encapsulation. After the gel formation, 500 μL
media (EBM supplemented with 2% FBS and soluble growth factors at
noted concentrations) were added to each well followed by incubation at
37 °C. The spheroids were imaged after 24 h, and at least four spheroids
were studied under each set of conditions.
Endothelial Cells Cultured on 2D Collagen Coating: A collagen solution
was prepared by mixing rat-tail type I collagen with PBS to give a final
collagen concentration of 0.1 mg mL−1 . For plain collagen coating,
100 μL of the solution was added at room temperature to the wells of a
96-well tissue-culture plate and incubated at 37 °C for 1 h. For peptide-
modified collagen coating, a stock solution of the peptide (1 mM)
was first heated at 80 °C for at least 30 mins to enable melting. This
heated peptide solution was then added directly to the collagen coating
solution at 1:10 and 1:40 dilutions (by volume), to create final peptide
concentrations of 100 μM and 25 μM, respectively. No quenching was
required since the heated peptides were quickly cooled during the
mixing step due to the large volume of the collagen coating solution.
The mixture solution (100 μL) was added to the wells of a 96-well plate
and incubated for 1 h. After the incubation, all excess solutions were
removed from the cell-culture well. Subsequently, 15,000 HUVECs
in 100 μL of endothelial basal media (EBM) supplemented with 2%
FBS were seeded in each well (96-well plate). For VEGF-supplemented
experiments, the 100 μL of growth media were further supplemented
with 500 ng mL−1 VEGF.
ERK Assay: Cells were first serum-starved in reduced serum media
(EBM supplemented with 0.25% FBS) for 4 h. For plain collagen
coating, 1.5 mL of the collagen solution (0.1 mg mL−1 ) was added at
room temperature to the wells of a six-well tissue culture plate and
incubated at 37 °C for 1 h. For peptide-modified collagen coating, a
stock solution of the QKCMP peptide (1 mM) was first heated at 80 °C
for at least 30 min to enable melting. This heated peptide solution was
then added directly to the collagen coating solution at 1:10 dilution (by
volume), to create a final peptide concentration of 100 μM. The QKCMP-
collagen solutions (1.5 mL) were then added to wells of the same six-
well tissue culture plate. The plate was incubated at 37 °C for 1 h, and
the remaining coating solution was removed. Each well was then seeded
with the serum-starved HUVECs (485,000 cells well−1 ) in 1.5 mL EBM
media supplemented with 2% FBS. As a positive control, cells seeded
on one of the plain collagen-coated wells were supplemented with
100 ng mL−1 soluble VEGF. The cells were first cultured for 1 h at 37 °C
and then lysed with lysis buffer (25 mM Tris-HCL, 0.5% Triton X-100,
200 mM NaCl, 2 mM EDTA, phosphatase and protease inhibitors; Roche
Applied Science). The cell lysate was spun at 15,000 g for 15 min at 4 °C,
and the supernatant was collected. Protein concentration within the
supernatant was determined using the Pierce BCA assay kit (Thermo
Fisher Scientific, Rockford, IL). The lysate (17 μg) was loaded into a
NuPAGE Novex 10% Bis-Tris gel (Invitrogen) for protein separation by
Patterned Collagen Substrate for Local EC Activation: A collagen
solution (110 μL, 0.1 mg mL−1 ) was added to individual wells of a
48-well plate with the plate tilted at a 45° angle so that only half the
surface area of each well was covered with the collagen solution. The
plate was then incubated at 37 °C in the tilted position for 1 h. After
removing the remaining collagen solution, the plate was tilted in the
opposite direction, followed by addition of QKCMP–collagen mixture
solution (110 μL, 100 μM QKCMP in 0.1 mg mL−1 collagen) previously
prepared by adding 80 °C QKCMP (11 μL, 1 mM) to type I collagen
solution (99 μL, 0.1 mg mL−1 ). The tilted plate was then incubated at
37 °C for 1 h, and the remaining coating solution was removed. The well
was then seeded with HUVECs (55,000 cells well−1 ) in 500 μL of EBM
media supplemented with 2% FBS and VEGF (500 ng mL−1 ).
Immunohistochemical Staining: The cells were fixed with 200 μL of
10% neutral buffered formalin for 30 min. After fixing, the cells were
permeabilized and blocked with 200 μL of TBS supplemented with 5%
donkey serum and 0.25% Triton X-100. Primary mouse anti-human
PECAM-1 antibodies (0.5 mg mL−1 ) were added at 1:50 dilution in TBS
and the mixture was incubated at 4 °C overnight. Subsequently, Cy3
conjugated anti-mouse secondary antibodies (0.5 mg mL−1 ) were added
at 1:250 dilution in TBS and the mixture was further incubated at room
temperature for 90 mins. Finally, the cells were stained with a 200 μL
PBS solution containing phalloidin (0.165 μM) and DAPI (1 μg mL−1 ) for
30 mins. The fluorescent cell images were captured on Nikon Eclipse
TE2000-E (Nikon Instruments, Melville, NY).
Supporting Information
Supporting Information is available from the Wiley Online Library or
from the author.
Acknowledgements
The authors thank Kalina Hristova and Edwin Li for help with circular
dichroism, Christopher Chen, Xingyu Liu, Jacob Koskimaki, and
Colette Shen for help with cell studies, J. Michael McCaffery and Erin
Pryce for help with electron microscopy, and Fenghao Chen, Lijuan
He, and Sarvenaz Sarabipour for help with Western blots. This work
was supported by grants from NSF (DMR-0645411) and NIAMS/NIH
©
wileyonlinelibrary.com
Adv. Funct. Mater. 2011, 21, 4252–4262
2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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(R01AR060484) to S.M.Y., by NSF IGERT fellowship to T.R.C., and by
HHMI Graduate Training Program (NBMed) and NDSEG (32 CFR 168a)
fellowships to P.J.S.
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