U.S. patent application number 16/743574 was filed with the patent office on 2020-07-16 for novel peptides for supporting endothelial progenitor cell rolling and capture and endothelialization of biomaterials.
The applicant listed for this patent is AUBURN UNIVERSITY. Invention is credited to ELIZABETH A. LIPKE, WEN JUN SEETO.
Application Number | 20200222596 16/743574 |
Document ID | / |
Family ID | 57516574 |
Filed Date | 2020-07-16 |
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United States Patent
Application |
20200222596 |
Kind Code |
A1 |
LIPKE; ELIZABETH A. ; et
al. |
July 16, 2020 |
NOVEL PEPTIDES FOR SUPPORTING ENDOTHELIAL PROGENITOR CELL ROLLING
AND CAPTURE AND ENDOTHELIALIZATION OF BIOMATERIALS
Abstract
The present invention relates to the production of
endothelialized matrices and materials from immature endothelial
cells using substrates to which particular peptides have been
grafted. The resultant substrates can be used to capture and
support immature endothelial cells. Further, the methods and
compositions of the present invention provide viable cell delivery
platforms that allow for production and provision of
endothelialized medical devices and implants, including vascular
grafts, stents, shunts, and valves, endothelialized surfaces and
channels for in vitro testing devices, including microfluidic
chips, and materials that support vascularization such as for use
in engineered tissues. The present invention includes novel methods
required for the successful production of cellularized substrates,
systems and components used for the same, and methods of using the
resultant cell and tissue compositions.
Inventors: |
LIPKE; ELIZABETH A.;
(Auburn, AL) ; SEETO; WEN JUN; (AUBURN,
AL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
AUBURN UNIVERSITY |
AUBURN |
AL |
US |
|
|
Family ID: |
57516574 |
Appl. No.: |
16/743574 |
Filed: |
January 15, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15099582 |
Apr 14, 2016 |
10568992 |
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16743574 |
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62147215 |
Apr 14, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 5/069 20130101;
A61L 27/227 20130101; A61L 31/047 20130101; C12N 2533/50 20130101;
C12N 2533/30 20130101; A61L 27/3683 20130101; A61L 31/005 20130101;
A61L 31/16 20130101 |
International
Class: |
A61L 31/16 20060101
A61L031/16; C12N 5/071 20060101 C12N005/071; A61L 31/04 20060101
A61L031/04; A61L 27/22 20060101 A61L027/22; A61L 27/36 20060101
A61L027/36; A61L 31/00 20060101 A61L031/00 |
Goverment Interests
GRANT REFERENCE
[0002] This invention was made with government support under
NSF-CBET-1150854 awarded by National Science Foundation, and
14SDG18610002 awarded by the American Heart Association. The
government has certain rights in the invention.
Claims
1-19. (canceled)
20. A method of capturing endothelial cells from a flowing fluid,
the method comprising: providing a substrate that is operably
linked to at least one first peptide comprising SEQ ID NO: 1 and at
least one second peptide comprising SEQ ID NO: 2; and exposing the
substrate to a source of endothelial cells in a flowing fluid.
21. The method of claim 20, wherein the endothelial cells are
immature endothelial cells, endothelial progenitor cells, or stem
cell-derived endothelial cells.
22. The method of claim 20, wherein the substrate is further
operably linked to at least one additional peptide selected from
the group consisting of SEQ ID NOs: 8, 11 and 18 and combinations
thereof.
23. The method of claim 20, wherein providing a substrate comprises
providing a medical device or implant.
24. The method of claim 23, wherein providing a medical device or
implant comprises providing a vascular graft, stent, shunt, or
valve.
25. The method of claim 20, wherein providing a substrate comprises
providing an in vitro testing device.
26. The method of claim 20, wherein the substrate comprises a
hydrogel.
27. The method of claim 20, wherein the substrate comprises a
polymer.
28. The method of claim 20, wherein the substrate comprises
biomimetic material.
29. The method of claim 28, wherein the substrate comprises
decellularized extracellular matrix (dECM).
30. The method of claim 20, wherein the molar ratio of the second
peptide to the first peptide is between 3:1 and 1:3.
31. The method of claim 20, wherein the flowing fluid is blood.
32. A composition for capturing endothelial cells from a flowing
fluid, the composition comprising: a substrate; at least one first
peptide comprising SEQ ID NO: 1 operatively linked to the surface
of the substrate; and at least one second peptide comprising SEQ ID
NO: 2 operatively linked to the surface of the substrate; wherein
the composition facilitates the capture of endothelial cells from a
flowing fluid.
33. The composition of claim 32, further comprising at least one
additional peptide selected from the group consisting of SEQ ID
NOs: 8, 11 and 18 and combinations thereof.
34. The composition of claim 32, wherein the endothelial cells are
immature endothelial cells, endothelial progenitor cells, or
pluripotent stem cell-derived endothelial cells.
35. The composition of claim 32, wherein the substrate comprises a
polymer.
36. The composition of claim 32, wherein said substrate comprises a
hydrogel.
37. The composition of claim 32, wherein said substrate comprises a
biomimetic material.
38. The composition of claim 37, wherein the substrate comprises
decellularized extracellular matrix (dECM).
39. The composition of claim 32, wherein the molar ratio of the
second peptide to the first peptide is between 3:1 and 1:3.
40. A composition for capturing endothelial cells from a flowing
fluid, the composition comprising: a substrate; at least one first
peptide comprising SEQ ID NO: 8 operatively linked to the surface
of the substrate; and at least one second peptide comprising SEQ ID
NO: 2 operatively linked to the surface of the substrate; wherein
the composition facilitates the capture of endothelial cells from a
flowing fluid.
41. A method of capturing endothelial cells from a flowing fluid,
the method comprising: providing the composition of claim 40; and
exposing the substrate to a source of endothelial cells in a
flowing fluid.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 15/099,582, filed Apr. 14, 2016, which claims priority under 35
U.S.C. .sctn. 119 to provisional application Ser. No. 62/147,215
filed Apr. 14, 2015, each are incorporated by reference in their
entireties.
FIELD OF THE INVENTION
[0003] The present invention is directed to compositions and
methods for the capture and support of immature endothelial cells.
More particularly, the invention is directed to endothelialization
of a substrate, including medical devices and implants. The capture
and support of immature endothelial cells relies on the presence of
two or more peptides or proteins, as described herein, grafted to
the surface of the substrate.
BACKGROUND OF THE INVENTION
[0004] Endothelial progenitor cells (EPCs) and pluripotent stem
cell-derived endothelial cells have the potential to become a
reliable source of autologous cells for endothelialization of
intravascular devices and vascularization of tissue engineered
constructs. In order to design biomaterials that can employ EPCs to
enhance endothelialization, however, a better understanding of
their dynamic adhesion to material surfaces under physiological
shear is needed.
[0005] Endothelial colony forming cells (ECFCs) are one type of
EPCs; ECFCs are highly proliferative and are capable of forming
mature and functional endothelial cells for vessel repair and
postnatal angiogenesis. EPCs are a subpopulation of monocytes that
are derived from myeloid cells, which are one type of leukocyte.
EPCs that have been isolated from blood and expanded in vitro are
frequently called late outgrowth or endothelial colony forming
cells (ECFCs). Expression of surface receptors on these cells
differs from other types of monocytes, "early outgrowth" EPCs, and
mature endothelial cells. Advantages of ECFCs for tissue
engineering applications include the relative ease and lack of
comorbidity in obtaining autologous cells, the highly proliferative
nature of ECFCs, and the ability of ECFCs to yield mature
endothelial cells. To exploit their potential, however, it is
necessary to first understand whether ECFCs behave similarly to ECs
in their abilities to interact with engineered biomimetic materials
and which cell surface receptors mediate these interactions.
[0006] As a precursor for endothelial cells (ECs), EPCs show an
endothelial-like phenotype with high proliferative capability
(especially late EPCs), and can be differentiated into mature
endothelial cells that form the endothelium. Hence, potential
clinical applications of EPCs include vessel repair,
neovascularization of ischemic organs, and coating of vascular
grafts. Previous work has shown that .beta.2 integrins are
important in EPC homing to sites of ischemia and
neovascularization. Integrins also play an important role in EPC
capture on EC or ECM proteins in vitro. Although the types and
distribution of integrin receptors are not well identified on EPCs,
prior work has estimated the number of .alpha..sub.5.beta..sub.1
and .alpha..sub.v.beta..sub.3 integrin receptors present on
umbilical cord-blood derived EPC using flow cytometry. Besides flow
cytometry, investigation of EPC migration has shown that blocking
.alpha..sub.5.beta..sub.1 decreases EPC migration. However,
understanding of the constitution and function of integrins present
on the membrane surface of EPCs is still very limited and needs to
be further explored. Importantly, although it was known that ECFCs
express .alpha..sub.5.beta..sub.1 and .alpha..sub.v.beta..sub.3
integrins, ECFCs were not known to express an integrin that would
bind REDV (SEQ ID NO:2) (discussed below).
[0007] Cardiovascular disease is one of many conditions that may be
treated by the insertion of stents and/or vascular grafts. However,
incomplete endothelialization can reduce the effectiveness of this
type of treatment. Therefore, the incorporation of endothelium
specific factors, for example, tailoring biomaterials for
cardiovascular implant coatings, will provide enhanced clinical
treatment alternatives. One such coating includes a nanofibrous
matrix that may be applied to the medical implant as a
self-assembled coating. Other such materials include biomimetic
materials and polymer coatings for implants and medical
devices.
[0008] Previous work has attempted to identify and characterize
peptides, including REDV (SEQ ID NO:2), RGDS (SEQ ID NO:3), and
YIGSRG (SEQ ID NO:4), grafted on PEG hydrogels have been shown to
support EPC rolling under shear. Despite the fact that REDV (SEQ ID
NO:2)-grafted hydrogels reduced EPC rolling velocity the most,
however, it does not support firm adhesion even at low shear rate.
Thus, there is a continuing need for peptides that are capable of
slowing and capturing EPCs. Notably, REDV (SEQ ID NO:2) (which
binds .alpha..sub.4.beta..sub.1), does not bind either
.alpha..sub.5.beta..sub.1 and .alpha..sub.v.beta..sub.3 integrin,
which are expressed on EPCs and pluripotent stem cell-derived
endothelial cells.
CRRETAWAC (SEQ ID NO:1)
[0009] The peptide CRRETAWAC (SEQ ID NO:1) has a high binding
affinity and selectivity for integrin .alpha..sub.5.beta..sub.1.
Prior work involved constructing a heptapeptide library by ligating
a synthetic oligonucleotide into fUSE 5 vector. The oligonucleotide
has a core sequence of TGT(NNK)7TGT (SEQ ID NO:6) where N
represents an equal molar mixture of A, C, G, T while K represents
G or T. TGT was coded for cysteine and NNK was coded for all amino
acid. The cysteines on each side of the peptide were designed to
form disulfide bond and to form cyclic peptides. Thus, the
oligonucleotide produces a library of cyclic peptides with seven
random amino acids. When the peptide library was screened with
.alpha.5.beta.1 coated wells to identify the peptide with high
binding affinity with .alpha.5.beta.1, among the non-RGD containing
peptides, CRRETAWAC (SEQ ID NO:1) showed the highest affinity to
.alpha.5.beta.1.
[0010] Other previous works has investigated CRRETAWAC (SEQ ID
NO:1) in endothelialization of expanded polytetrafluoroethylene
(ePTFE) surfaces by incorporating GSSSCRRETAWAC (SEQ ID NO:7) (FIG.
4). When ePTFE was modified with GSSSCRRETAWAC (SEQ ID NO:7), it
supported EC attachment and proliferation under static conditions.
Furthermore, a significant lower coverage of the surface by
platelets was observed comparing to RGD surface and FN-coated
glass. More notably, use of this peptide for capture of cells from
physiological flow conditions has not previously been
considered.
(C16)2-Glu-C2-KSSPHSRNSGSGSGSGSGRGDSP (SEQ ID NO:8) (PR_b) for
.alpha.5.beta.1
[0011] Despite the fact that PRb contained the ubiquitous RGD
peptide and the synergistic PHSRN (SEQ ID NO:9) peptide, this
peptide is chosen to test for the capability on EPC rolling due to
the its superior design in accurately mimicking FN's binding
affinity for .alpha..sub.5.beta..sub.1. This peptide has been shown
to mimic FN through HUVEC adhesion, spreading, and extracellular FN
production (Mardilovich et al., 2006). In native FN, the distance
between PHSRN (SEQ ID NO:9) and RGD is 30-40 .ANG. which is a
critical factor for PHSRN (SEQ ID NO:9) to perform its synergistic
role in adhesion. With the length for each amino acid being 3.7
.ANG., the Kokkoli Lab used SGSGSGSGSG (SEQ ID NO:10), which is a
total of 10 amino acids to give a linker distance of 37 .ANG., to
match the distance between PHSRN (SEQ ID NO:9) and RGD. In
addition, it has been shown that the ratio of hydrophilic to
hydrophobic residues in between PHSRN (SEQ ID NO:9) and RGD in FN
is almost 1:1 (Mardilovich & Kokkoli, 2004). The repeating SG
sequence was chosen to mimic this ratio. Therefore, PRb was able to
mimic the adhesion property of FN for .alpha.5.beta.1.
HSDVHK (SEQ ID NO:11) (P11)
[0012] P11 was discovered by screening through PS-SPCL using
protein-protein competitive inhibition assay. The PS-SPCL that was
studied was comprised of 114 types of hexapeptide mixture and this
library was divided into 6 groups with each group has various amino
acids residues at each position. The PS-SPCL together with
fluorescently labeled vitronectin (VN) were added onto
.alpha..sub.v.beta..sub.3-coated surface and allowed to compete for
the .alpha..sub.v.beta..sub.3 integrin. As shown in FIG. 5,
peptides with histidine, H, at Position 1 showed the lowest
fluorescent intensity meaning it was highly competitive for
.alpha..sub.v.beta..sub.3 and inhibited the fluorescently labeled
VN to bind .alpha..sub.v.beta..sub.3. Similarly, histidine at
Position 5 and lysine, K, at position 6 showed the same result. On
the other hand, more than one amino acid showed similar result at
Position 2, 3, and 4. Therefore, 12 hexapeptides with sequence
HXXXHK (SEQ ID NO:13) were synthesized where X represents
glycine/histidine/serine at Position 2, aspartic acid/leucine at
Position 3, and leucine/valine at Position 4. These 12 hexapeptides
were further screened using the same competitive inhibition assay
and HSDVHK (SEQ ID NO:11) was found to be the most competitive for
.alpha..sub.v.beta..sub.3. Thus, HSDVHK (SEQ ID NO:11) has a high
specificity and affinity for .alpha..sub.v.beta..sub.3 and it has
high potential in capturing .alpha..sub.v.beta..sub.3-expressing
EPCs.
NCKHQCTCIDGAVGCIPLCP (SEQ ID NO:12) (V2)
[0013] V2 is a peptide representing the 116-135 residues of the
cysteine-rich heparin binding protein (CCN1). The functions of CCN1
include regulating cell adhesion, migration, proliferation,
survival and differentiation in mesenchymal cells. Studies have
shown that CCN1 binds directly to .alpha..sub.v.beta..sub.3 and
mediates pro-angiogenic activities. Previous studies have evaluated
peptides of different portion of CCN1 and reported that V2 was
responsible portion for HUVEC adhesion specifically through
.alpha..sub.v.beta..sub.3. Previous work has also shown that when
the D residue was altered into A, the altered V2 peptide lost the
capability to bind HUVEC through .alpha..sub.v.beta..sub.3
integrin. Furthermore, shorter versions of V2 have also shown
similar results meaning V2 is the exact peptide required to bind
.alpha..sub.v.beta..sub.3. Therefore, V2 is a potential peptide to
capture EPCs due to its specific binding to
.alpha..sub.v.beta..sub.3.
BRIEF SUMMARY OF THE INVENTION
[0014] The present invention relates to previously unknown peptides
and combinations of peptides that significantly increase the
capture rate of endothelial cells, including endothelial colony
forming cells (ECFCs) under flow conditions with physiological
shear rates. The endothelial cell capturing peptides may include
CRRETAWAC (SEQ ID NO:1) and PRb (SEQ ID NO: 8). The combination may
further involve an endothelial cell slowing peptide REDV (SEQ ID
NO:2) and an endothelial cell capturing peptide CRRETAWAC (SEQ ID
NO:1). REDV (SEQ ID NO:2) has shown to significantly decrease ECFC
rolling velocity compared to other bioactive peptides, including
RGDS (SEQ ID NO:3), YIGSRG (SEQ ID NO:4), P11 (SEQ ID NO: 11), PRb
(SEQ ID NO: 8), and CRRETAWAC (SEQ ID NO:1). Despite the fact that
REDV (SEQ ID NO:2)-grafted hydrogels had the greatest reduction in
EPC rolling velocity, REDV (SEQ ID NO:2) does not support capture
even at low shear rates. Surprisingly, however, cell capture events
were consistently observed on CRRETAWAC (SEQ ID NO:1)-grafted
hydrogels and on PRb (SEQ ID NO: 8), grafted hydgrogels at 20
s.sup.-1. In our analysis, 3.74%.+-.1.0% of ECFCs were captured on
CRRETAWAC (SEQ ID NO:1)-grafted hydrogels and 1.14%.+-.0.2% of
ECFCs were captured on PRb grafted hydrogels. To further evaluate
the potential use of CRRETAWAC (SEQ ID NO:1) in the ECFC capture
for endothelialization, ECFC rolling on hydrogels that were grafted
with combinations of CRRETAWAC (SEQ ID NO:1) with REDV (SEQ ID
NO:2), which has a high affinity for the integren
.alpha..sub.4.beta..sub.1, were assessed. Hydrogels grafted with
REDV (SEQ ID NO:2)/CRRETAWAC (SEQ ID NO:1) combination (0.35
.mu.mol/.mu.L of REDV (SEQ ID NO:2)/0.35 pmol/ml of CRRETAWAC (SEQ
ID NO:1)) had significantly increased ECFC capture of 13.4%.+-.2.3%
as compared to CRRETAWAC (SEQ ID NO:1) alone. Thus, we have
discovered the peptides CRRETAWAC (SEQ ID NO:1) and PRb and the
combination of CRRETAWC (SEQ ID NO:1)/REDV (SEQ ID NO:2) for
capturing ECFCs under shear including potential use in establishing
or recovering the vascular endothelium.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] The following drawings form part of the specification and
are included to further demonstrate certain embodiments or various
aspects of the invention. In some instances, embodiments of the
invention can be best understood by referring to the accompanying
drawings in combination with the detailed description presented
herein. The description and accompanying drawings may highlight a
certain specific example, or a certain aspect of the invention.
However, one skilled in the art will understand that portions of
the example or aspect may be used in combination with other
examples or aspects of the invention.
[0016] FIG. 1 shows ECFC rolling velocities on peptide-grafted PEG
hydrogels. REDV (SEQ ID NO:2)-grafted hydrogels had significantly
lower ECFC rolling velocities. Means that do not share the same
letter are significantly different (p<0.05).
[0017] FIG. 2 shows ECFC captured rate on CRRETAWAC (SEQ ID NO:1),
PRb (SEQ ID NO: 8), and with REDV (SEQ ID NO:2) combinations.
[0018] FIG. 3 (A-F) shows inhibition of integrin
.alpha..sub.5.beta..sub.1-FN interaction by a hexapeptide PS-SPCL.
Generally, the y-axis represents the relative fluorescence
intensity of fluorescently-labeled FN that was bound to the
integrin .alpha..sub.5.beta..sub.1 coated ProteoChip, while the
x-axis represents the peptides with different amino acids at the
corresponding position. The amino acids at each position of the
hexapeptide that substantially inhibited the binding of the
fluorescently labeled FN to .alpha..sub.5.beta..sub.1 are
highlighted by boxes. As indicated on each sub-figure the lower the
relative fluorescence intensity, the higher the binding affinity of
the peptide to .alpha..sub.5.beta..sub.1. FIG. 3A illustrates
results for a peptide with an amino acid located at Position 1.
FIG. 3B illustrates results for a peptide with an amino acid
located at Position 2. FIG. 3C illustrates results for a peptide
with an amino acid located at Position 3. FIG. 3D illustrates
results for a peptide with an amino acid located at Position 4.
FIG. 3E illustrates results for a peptide with an amino acid
located at Position 5. FIG. 3F illustrates results for a peptide
with an amino acid located at Position 6.
[0019] FIG. 4 shows the structure of CRRETAWAC (SEQ ID
NO:1)-containing cyclic peptide for use in modification of ePTFE.
This peptide allowed high EC binding through
.alpha..sub.5.beta..sub.1 integrin and showed low platelet
adhesion.
[0020] FIG. 5 (A-F) shows inhibition of integrin
.alpha..sub.v.beta..sub.3-VN by a hexapeptide PS-SPCL. Generally,
the y-axis represents the relative fluorescence intensity of
fluorescently-labeled VN bound to the integrin
.alpha..sub.v.beta..sub.3 coated ProteoChip, while the x-axis
represents the peptides with different amino acids at the
corresponding position. The amino acids at each position of the
hexapeptide that substantially inhibited the binding of the
fluorescently labeled VN to .alpha..sub.v.beta..sub.3 are
highlighted by boxes. FIG. 5A illustrates results for a peptide
with an amino acid located at Position 1. FIG. 5B illustrates
results for a peptide with an amino acid located at Position 2.
FIG. 5C illustrates results for a peptide with an amino acid
located at Position 3. FIG. 5D illustrates results for a peptide
with an amino acid located at Position 4. FIG. 5E illustrates
results for a peptide with an amino acid located at Position 5.
FIG. 5F illustrates results for a peptide with an amino acid
located at Position 6.
[0021] FIG. 6 (A-C) shows adhesion of ECFCs on novel
peptides-grafted PEG hydrogels after 24 hours. ECFC adhesion on (A)
CRRETAWAC (SEQ ID NO:1)-(B) PRb (SEQ ID NO: 8), -(C) P11 (SEQ ID
NO: 11)-grafted PEG hydrogels.
[0022] FIG. 7 (A-B) shows ECFC rolling velocity between novel
peptides and known ECM peptides. As each shear rate, conditions
that do not share the same letter are significantly different
(p<0.05) from each other based on Tukey's test. Data represent
mean.+-.SD (n=3). FIG. 7A provides a graphical representation of
shear rate, while FIG. 7B provides the corresponding data in table
format.
[0023] FIG. 8 shows the distribution of rolling velocities for
rolled ECFCs on the peptide-grafted hydrogels at each tested shear
rate. Each marker represents one tracked cell. Separately prepared
peptide-grafted hydrogels were used for each trial.
[0024] FIG. 9 shows the number of ECFC captured between novel
peptides and known ECM peptides. Conditions that do not share the
same letter are significantly different (p<0.05) based on
Tukey's test. Data represent mean.+-.SD (n=3).
[0025] FIG. 10 shows rolling velocity between novel peptides, known
ECM peptides, and combinations thereof. Conditions that do not
share the same letter are significantly different (p<0.05) from
Tukey's test. Data represent mean.+-.SD (n=3).
[0026] FIG. 11 shows the distribution of rolling velocities for
rolled ECFCs on the peptide-grafted hydrogels at each tested shear
rate. Each marker represents one tracked cell. Separately prepared
peptide-grafted hydrogels were used for each trial.
[0027] FIG. 12 shows a comparison between calculated fluid
velocities and ECFC velocity. The velocity of ECFCs on the surface
of PEG hydrogels was less than the calculated fluid velocity at
approximately one cell diameter (15 .mu.m) and greater than the
calculated fluid velocity at approximately one cell radius (7.5
.mu.m) above the hydrogel surface. Data represent mean.+-.SD (n=3).
Therefore, to eliminate any ECFCs that might be non-specifically
interacting with the PEG hydrogel surface, the cutoff for
identification of rolling cells was set at 40% of the average fluid
velocity.
[0028] FIG. 13 (A-C) shows grafting PEG hydrogels with RGDS (SEQ ID
NO:3) significantly slowed ECFC velocity. (A) The ECFCs velocity on
RGES (SEQ ID NO:5)-grafted hydrogels was similar to the velocity on
ungrafted PEGDA hydrogels, while ECFC rolling velocity on RGDS (SEQ
ID NO:3)-grafted hydrogel showed a significant decrease compared to
RGES (SEQ ID NO:5)-grafted and PEGDA hydrogels at all shear rates.
Data represent mean.+-.SD (n=3). (*p<0.05 between RGDS (SEQ ID
NO:3)-grafted hydrogels and ungrafted PEGDA hydrogels. +p<0.05
between RGDS (SEQ ID NO:3)- and RGES (SEQ ID NO:5)-grafted
hydrogels). (B,C) Instantaneous velocity, average rolling velocity
and 40% of average fluid velocity of representative ECFCs on PEG,
PEG-RGES (SEQ ID NO:5), and PEG-RGDS (SEQ ID NO:3) hydrogels at
shear rates of 80 s.sup.-1 and 120 s.sup.-1, respectively.
[0029] FIG. 14 (A-C) shows a series of extracted frames following a
representative ECFC through the flow chamber on (A) PEG, (B) 5.6
.mu.mol/mL RGES (SEQ ID NO:5)-grafted, (C) 0.7 .mu.mol/mL RGDS (SEQ
ID NO:3)-grafted hydrogels. Scale bar=30 .mu.m.
[0030] FIG. 15 (A-C) shows a comparison of ECFC rolling on REDV
(SEQ ID NO:2)-, RGDS (SEQ ID NO:3)-, and YIGSRG (SEQ ID
NO:4)-grafted hydrogels. A) ECFC rolling velocity was lower on REDV
(SEQ ID NO:2)-grafted hydrogels as compared to RGDS (SEQ ID
NO:3)-grafted hydrogels and YIGSRG (SEQ ID NO:4)-grafted hydrogels
at all shear rates. Data represent mean.+-.SD (n=3). (* and
+p<0.05 within groups of 80 s.sup.-1 and 120 s.sup.-1). B,C)
Instantaneous velocity, average rolling velocity and 40% of average
fluid velocity of representative ECFCs on REDV (SEQ ID NO:2)-, RGDS
(SEQ ID NO:3)-, and YIGSRG (SEQ ID NO:4)-grafted hydrogels at 80
s.sup.-1 and 120 s.sup.-1, respectively.
[0031] FIG. 16 (A-C) shows a series of extracted frames following a
representative ECFC through the flow chamber on (A) REDV (SEQ ID
NO:2)-, (B) RGDS (SEQ ID NO:3)-, and (C) YIGSRG (SEQ ID
NO:4)-grafted hydrogels. Scale bar=30 .mu.m.
[0032] FIG. 17 shows the distribution of rolling velocities for
tracked ECFCs on the peptide-grafted hydrogels at each tested shear
rate. Each marker represents one tracked cell. Separately prepared
peptide-grafted hydrogels were used for each trial. At shear rates
of 80 s.sup.-1 and 120 s.sup.-1, more cells are grouped at the
lower rolling velocities for REDV (SEQ ID NO:2)-grafted hydrogels
than for RGDS (SEQ ID NO:3)- and YIGSRG (SEQ ID NO:4)-grafted
hydrogels.
[0033] Various embodiments of the present invention will be
described in detail with reference to the drawings, wherein like
reference numerals represent like parts throughout the several
views. Reference to various embodiments does not limit the scope of
the invention. Figures represented herein are not limitations to
the various embodiments according to the invention and are
presented for exemplary illustration of the invention.
DETAILED DESCRIPTION OF THE INVENTION
[0034] The following definitions and introductory matters are
provided to facilitate an understanding of the present
invention.
[0035] Numeric ranges recited within the specification, including
ranges of "greater than," "at least," or "less than" a numeric
value, are inclusive of the numbers defining the range and include
each integer within the defined range.
[0036] The singular terms "a", "an", and "the" include plural
referents unless context clearly indicates otherwise. Similarly,
the word "or" is intended to include "and" unless the context
clearly indicate otherwise. The word "or" means any one member of a
particular list and also includes any combination of members of
that list.
[0037] "Biomimetic materials" refers to materials which act to
emulate properties from a natural biological environment. Such
materials may be selected based on one or more characteristics,
including, for example, ability to maintain the mechanical and
electrical properties of the native tissue, direct cell and tissue
orientation, deliver particular drugs and growth factors, and
degrade in response to enzymes secreted by cells. Furthermore,
these materials can be selected based on ability to promote cell
adhesion, mechanical stretch and electrical conduction. Overall,
biomimetic materials can be engineered or selected to produce
functional tissue with highly controlled, defined properties.
Examples of biomimetic materials include natural materials and
synthetic materials. Natural materials include, but are not limited
to, materials derived from proteins, polysaccharides, and other
derivatives of these substances, such as, for example, collagen,
gelatin, glycosaminoglycans (e.g. hyaluronic acid), elastin,
fibronectin, laminin, fibrin, and alginates. Synthetic materials
include temporally-changing or externally-modifiable materials that
can be engineered to provide biomimetic properties that facilitate
cardiac regeneration. Materials that fall into this category are
unique in their ability to change structure based on changes in
input (temperature, pH, photoactive, mechanical/electrical stress
hydrogels, environmentally-responsive polymers, and conductive
materials.
[0038] The term "crosslink" refers to a bond or chain of atoms
attached between and linking two different polymer chains.
[0039] "Endothelialized" and "endotheliazation" refer to formation
of a coating layer of endothelial cells. Endotheliazation may
include protecting underlying materials or large vessels that have
sustained damage, such as from blood flow, and may also include
prevention of restenosis by inhibiting overgrowth of other cell
types, particularly in the case of stents and other implants.
[0040] "Enriching," as the term is used herein, refers to the
process by which the concentration, number, or activity of
something is increased from a prior state. For example, a
population of 100 ECFCs is considered to be "enriched" in ECFCs if
the population previously contained only 50 ECFCs. Similarly, a
population of 100 ECFCs is also considered to be "enriched" in
ECFCs if the population previously contained 99 ECFCs. Likewise, a
population of 100 ECFCs is also considered to be "enriched" in
ECFCs even if the population previously contained zero ECFCs.
[0041] The terms "graft" and "grafting" generally refer to the
attachment or conjugation of two or more materials. With respect to
the present invention, grafting refers to any process or method by
which a protein or peptide can be attached to a polymer, substrate,
matrix or material.
[0042] The term "hydrogel" refers to a water-swellable polymeric
matrix, consisting of a three-dimensional network of macromolecules
held together by covalent crosslinks that can absorb a substantial
amount of water to form an elastic gel.
[0043] As the term is used herein, "isolated" refers to a
polynucleotide, polypeptide, protein, molecule, compound, material
or cell of genomic or synthetic origin or some combination thereof
which is not associated with all or a portion of the
polynucleotides, polypeptides, proteins, molecules, compounds,
materials or cells with which the isolated polynucleotide,
polypeptide, protein, molecule, compound, material or cell is found
in nature, or is linked to a polynucleotide, polypeptide, protein,
molecule, compound, material or cell to which it is not linked in
nature.
[0044] "Maintenance" of a cell or a population of cells refers to
the condition in which a living cell or living cell population is
neither increasing nor decreasing in total number of cells in a
culture. Alternatively, "proliferation" of a cell or population of
cells, as the term is used herein, refers to the condition in which
the number of living cells increases as a function of time with
respect to the original number of cells in the culture.
[0045] The term "operably linked" as used herein refers to the
joining, coupling, or linking of a peptide to a surface through a
wide range of chemistries into an equally large number of different
materials. A peptide can be operably linked to a surface, for
example, by grafting, bonding, coating, linking, crosslinking,
polymerizing, co-polymerizing, or integrating. For example, one or
more peptides may be incorporated into a substrate material as the
substrate material is being formed, such as incorporation into a
nanofibrous matrix or a multilayer vascular graft.
[0046] The term "PEG" as used herein refers to poly(ethylene
glycol).
[0047] The phrase "pluripotent stem cells" (PSCs) refers to stem
cells that have the potential to differentiate into any of the
three germ layers: endoderm (interior stomach lining,
gastrointestinal tract, the lungs), mesoderm (muscle, bone, blood,
urogenital), or ectoderm (epidermal tissues and nervous system).
PSCs of the present invention include embryonic PSCs and induced
PSCs, which are derived from a non-pluripotent cell, typically an
adult somatic cell, generated by any method known in the art,
including, for example, through introduction or activation of
specific transcription factors and/or genes.
[0048] As the term is used herein, "population" refers to two or
more cells.
[0049] The term "physiological shear" refers to the frictional
force tangential to the direction of a flowing fluid, the force of
which is directly related to the fluid's viscosity shear stress. In
blood vessels, shear stress acts on endothelium and is the
mechanical force responsible for the acute changes in luminal
diameter.
[0050] A "somatic cell" is understood to be a biological cell
ordinarily found in a multicellular organism that is not a gamete,
germ cell, gametocyte, or undifferentiated stem cell. Somatic cells
include cells of the endoderm (interior stomach lining,
gastrointestinal tract, the lungs), mesoderm (muscle, bone, blood,
urogenital), and ectoderm (epidermal tissues and nervous
system).
[0051] "Substantially homogeneous," as the term is used herein,
refers to a population of a substance that is comprised primarily
of that substance, and one in which impurities have been
minimized.
[0052] "Vascularization" is the process of new blood vessel
formation, including vessel formation to support wound healing,
perfusion of engineered tissues.
Cellularization Compositions
[0053] In one aspect, the present invention involves compositions
for inducing rolling and stopping of immature endothelial cells. In
a further aspect, the compositions are effective for
cellularization, specifically endothelialization and
vascularization. In one aspect, the compositions comprise a
substrate to which is grafted two or more peptides or proteins that
mediate rolling, capture, and support of immature endothelial cells
under physiological shear.
[0054] Peptides and proteins suitable for the compositions and
methods of the present invention are generally adhesion ligand
peptides and peptides capable of binding integrins. In a further
aspect, the proteins and peptides are capable of binding to
.alpha..sub.5.beta..sub.1, .alpha..sub.4.beta..sub.1, or
.alpha..sub.v.beta..sub.3 cell surface integrin. In one embodiment,
the proteins or peptides comprise one or more proteins or peptides
capable of binding to .alpha..sub.5.beta..sub.1 or
.alpha..sub.v.beta..sub.3 cell surface integrin, and one or more
proteins or peptides capable of binding .alpha..sub.4.beta..sub.1.
In another embodiment, the proteins or peptides are selected from
the group consisting of CRRETAWAC (SEQ ID NO:1), YIGSRG (SEQ ID
NO:4), REDV (SEQ ID NO:2), RGDS (SEQ ID NO:3), P11 (SEQ ID NO: 11),
and PRb (SEQ ID NO: 8). In an even more preferred embodiment, the
compositions and methods of the present invention involve two or
more peptides, most preferably comprising CRRETAWAC (SEQ ID NO:1)
and REDV (SEQ ID NO:2) or PRb (SEQ ID NO: 8), and REDV (SEQ ID
NO:2). In a further aspect, proteins and peptides of the invention
include proteins and peptides that incorporate any of CRRETAWAC
(SEQ ID NO:1), YIGSRG (SEQ ID NO:4), REDV (SEQ ID NO:2), RGDS (SEQ
ID NO:3), P11 (SEQ ID NO: 11), and PRb (SEQ ID NO: 8).
[0055] According to one aspect of the invention, the compositions
and methods include a substrate (i.e. a surface, matrix or
material) operably linked to one or more of the peptides described
herein. In one further aspect, the substrate can be a polymer
compatible with cellular vascularization. In another aspect, the
substrate is a biomimetic matrix or material, including materials
suitable for use in medical devices or implants, including but not
limited to vascular grafts.
[0056] In one aspect, the substrate can be a synthetic
polymer-based material, such as expanded poly(tetrafluoroethylene)
and poly-(ethylene terephthalate) (Dacron). In another aspect,
biomimetic materials for use in the present invention include
biomaterials that can be engineered or selected to produce
functional tissue with highly controlled, defined properties.
Examples of biomimetic materials include natural materials and
synthetic materials, or a combination of natural materials and
synthetic materials. Examples of biomaterials can be found in U.S.
Pat. No. 8,691,276, titled "Extracellular matrix-derived gels and
related methods" which is incorporated herein in its entirety.
Natural materials include, but are not limited to, materials
derived from proteins, polysaccharides, and other derivatives of
these substances, such as, for example, collagen, gelatin,
glycosaminoglycans (e.g. hyaluronic acid), elastin, fibronectin,
laminin, fibrin, and alginates. Synthetic materials include
temporally-changing or externally-modifiable materials that can be
engineered to provide biomimetic properties that facilitate cardiac
regeneration. Examples of biomimetic materials include, but are not
limited to, polycaprolactone (PCL), poly(glycolic acid) (PGA),
poly-4-hydroxybutyrate (P4HB), poly (lactic acid) (PLA) and
poly(lactic-co-glycolic acid) (PLGA), poly(glycerol-sebacate)
(PGS), poly(trimethylene carbonate-co-lactide), polyester urethane
urea (PEUU) 129, Pluronic F127/GelMA, HA/N-cadherin mimetic
peptides, MaHA/MMP-degradable crosslinker/BMP-2,
fibrin/PDGF-BB/BMP-2, PEGDMA, expanded polytetrafluoroethylene
(ePTFE), poly(ethylene terephthalate) (PET) and polyurethanes (PU),
biodegradable polymers such as poly(lactide-co-glycolide),
poly(ethylene glycol)-b-poly(L-lactide-coe-caprolactone) and
polydepsipeptides, polyurethane and combinations thereof; and
copolymers of natural and synthetic materials. Materials that fall
into this category are unique in their ability to change structure
based on changes in input (temperature, pH, photoactive,
mechanical/electrical stress hydrogels, environmentally-responsive
polymers, and conductive materials). Other artificial extracellular
matrix proteins that can be used for, or compose part of,
substrates of the present invention include recombinant
cellulose-binding domain (CBD)-RGD fusion protein, GFOGER.
[0057] In another aspect, the substrate comprises natural polymers
and biomimetic materials. Natural polymers include ECM-derived
collagen, fibronectin, laminin, hyaluronic acid, and vitronectin.
An exemplary natural or synthetic biomimetic material includes
hydrogels. Hydrogels are insoluble, cross-linked polymer network
structures composed of hydrophilic homo- or hetero-co-polymers,
which have the ability to absorb significant amounts of water.
Consequently, this is an essential property to achieve an
immunotolerant surface and matrix (i.e., with respect to protein
adsorption or cell adhesion). Due to their significant water
content, hydrogels also possess a degree of flexibility very
similar to natural tissue, which minimizes potential irritation to
surrounding membranes and tissues. In general, hydrogels are strong
hydrophiles, capable of supporting nutrient transport and
controlled degradation to allow for cell proliferation, generally
composed of self-assembling materials, namely peptide amphiphiles.
Hydrogels are highly absorbent (they can contain over 90% water)
natural or synthetic polymeric networks. Hydrogels also possess a
degree of flexibility very similar to natural tissue, due to their
significant water content. Hydrogels for use in the present
invention include both entangled hydrogels and covalently linkable
hydrogels. In a further aspect, covalently-linkable hydrogels are
used as the biomimetic material based on advantageous
characteristic, including, for example, the ability to reproducibly
form three-dimensional structures using molds or deposition
techniques. Covalently-linkable hydrogels include all aqueous-based
covalently-linkable hydrogels, including all hydrogels formed from
natural polymers and hydrogels formed from synthetic polymers.
Covalently linkable hydrogels also include PEG-based (polyethylene
glycol) and non-PEG-based polymers; non-PEG-based hydrogels include
both natural and synthetic hydrogels. In a most particular aspect,
hydrogels of the present invention can be composed of PEGylated
fibrinogen, where the naturally occurring component fibrinogen is
directly coupled to acrylated PEG to form PEG-fibrinogen. In
another aspect, the biomimetic material of the present invention
can be an acrylated gelatin.
[0058] In certain aspects, the substrate may comprise
decellularized extracellular matrix (dECM), such as decellularized
ovine pulmonary heart valve cusps (dPVCs) and decellularized rat
aortic grafts (dAoGs).
[0059] In one aspect, the methods of the present invention can
include modulation of the mechanical properties of a hydrogel for
different types of cells and/or tissues. In addition, the methods
can use state-of-the-art soluble factors and/or media formulations
to drive formation of a variety of somatic cells and tissues. In
one aspect, the methods can be used to generate cardiac cells or
tissues. In another aspect, for example, the methods can be used to
generate neural tissue formation, using similar PEG-fibrinogen
crosslinking density and/or mechanical properties as those used for
production of cardiac cells and tissues, but different soluble
factors and media formulation. The density and crosslinking
properties of PEG-fibrinogencan be varied by one or more of the
following: crosslinking time/light intensity, concentration of
PEG-fibrinogen, inclusion of a porogen (e.g. gelatin beads), or
addition of 1% or 2% PEG-diacrylate (with or without a matrix
metalloproteinase degradable peptide segment, such as
acryl-PEG-degradable_peptide-PEG-acryl).
[0060] The compositions of the present invention may comprise
additional elements that can aid in cell capture, cell support,
and/or cell maintenance. Additional components include additional
cell adhesion peptides, such as RGDS (SEQ ID NO:3) and YIGSRG (SEQ
ID NO:4), heparin, fibrinogen/fibrin, fibronectin,
collagen/gelatin, albumin, laminin, the tripeptide Arg-Gly-Asp
(RGD), stem cell factor (SCF; c-kit ligand), and CXCR2 ligand. The
compositions may also include additional peptides for binding
outgrowth endothelial cells, as described in U.S. Pat. No.
8,426,367.
[0061] In a further aspect of the invention, the peptides described
herein are operably linked to the surface of the substrate. In one
embodiment, the peptides can be operably linked to the substrate by
incorporation into the substrate material. In another aspect, the
peptides can be operably linked to the substrate through grafting,
including by any method known in the art, such as various physical,
chemical, and biofunctionalization techniques, which can also be
combined. For example, the grafting can be passive adsorption or
covalent grafting. Physical and chemical modification can be used
to influence protein and peptide adsorption. Proteins and peptides
can also be grafted to the substrate surface using
biofunctionality, including passive coating, covalent linking, and
presenting peptide linkers to sequence specific peptides from the
environment. Chemical modifications that may be used for coupling
proteins or peptides to substrates are summarized in Table 1.
TABLE-US-00001 TABLE 1 Peptide or protein Coupling or Substrate
surface reactive group cross-linking reagent functional group
--NH.sub.2 cyanogen bromide --OH cyanuric chloride DMT-MM --NH2--OH
glutaraldehyde --NH.sub.2 succinate anhydride diisocyanate
compounds diisothoncyanate compounds --NH2 nitrous acid --NH.sub.2
--SH --Ph--OH hydrazine and nitrous acid --COOH DMT-MM --NH.sub.2
carbodiimide compounds (EDC, DCC) --SH disulfide compound --SH
--NH2 thionyl chloride --COOH N-hydroxysuccinimide
N-hydroxysulfosuccinimide + EDC DCC, dicyclohexylcarbodiimide;
DMT-MM, 4(4,6-dimethoxy-1,3,5-triazin-2-yl)-4- methylmorpholinium
chloride; EDC, 1-ethyl-3 -(3-dimethylaminopropyl) carbodiimide
hydrochloride
Capture of Endothelial Cells and Support of Endothelial Cells Under
Physiological Shear
[0062] In another aspect, the present invention involves methods
for capture of immature endothelial cells and support of
endothelial cells under physiological shear. In one aspect, a
source of endothelial cells is exposed to a substrate that
comprises one or more integrin-binding peptides. The peptides can
be operably linked to the substrate, for example by grafting to the
surface of the substrate, coupling to the substrate, or
incorporation into the substrate. Sources of endothelial cells
include, immature endothelial cells, endothelial progenitor cells
embryonic stem cell-derived endothelial cells, pluripotent stem
cell-derived endothelial cells, induced pluripotent stem
cell-derived endothelial cells and adult stem cell sources, such as
adipose derived stem cells, mesenchymal stem cells, and bone marrow
derived stem cells. In a further aspect, sources of immature
endothelial cells may include a homogenous population or fraction
of cells, or may include a heterologous population of more than one
cell type or fraction of cells. The sources of immature endothelial
cells may primary endothelial cells, such as primary coronary
artery cells, primary aortic endothelial cells, primary pulmonary
artery endothelial cells, primary umbilical vein endothelial cells,
and primary dermal microvascular endothelial cells, or cell lines
such as SK-HEP-1 cells, HUVEC cells, TeloHAEC cells, EA.hy926
cells, TIME cells, NF.kappa.B-TIME cells, HMEC-1 cells, HULEC-5a
cells. Endothelial cells can be identified or characterized by gene
and protein expression (including, for example, CD31, CD105, CD144,
ZO-1, and von Willebrand Factor) and endothelial cell functions
(including, for example, tubular network formation, acetylated LDL
uptake, barrier function, and wound healing).
[0063] Sources of immature endothelial cells used in the present
invention can also include cells present in or isolated from a
subject. Samples that can be obtained from a subject for use in the
present invention include blood or blood parts (i.e. cell pellet or
cell fractions obtained by density gradient centrifugation), tissue
homogenates, or ex vivo expanded cell populations derived from a
sample. The samples can be enriched for immature endothelial cells,
for example, by fractionation, density gradient centrifugation,
flow cytometry, cell sorting, and the like.
[0064] In a further aspect, the methods involve capture of immature
endothelial cells by the peptides or proteins grafted to the
substrate. Capture is carried out by exposing the cells to the
substrate bearing the peptides under conditions that will permit
cell attachment to the substrate. In one embodiment, immature
endothelial cells are seeded directly onto the substrate. The cells
can be seeded at 1,000-100,000 cells/cm.sup.2, depending on the
source of endothelial cells. For example, a cell fraction obtained
from blood may require a higher seeding density, while a population
of cells obtained by primary cell culture may require a lower
seeding density, due to the relative numbers of immature
endothelial cells present in each source. In another aspect, the
exposure may occur under fluid flow conditions. In one embodiment,
the cells and substrate may be subjected to physiological
shear.
[0065] In another aspect, the present methods can be used to
capture immature endothelial cells in vivo. Thus, the source of
immature endothelial cells may be the blood of the subject in
vivo.
Populating a Substrate Surface With Peptides to Capture and Support
Immature Endothelial Cells
[0066] In another aspect, the present invention involves methods
for producing substrates or materials that selectively bind
endothelial progenitor cells. In a particular aspect, the methods
comprise providing a substrate. The substrate or material can be
composed of any substance that will support cell capture and
maintenance, including polymers, hydrogels, and other biomimetic
matrices and materials, as discussed herein. In one embodiment, the
substrate is a material suitable for a medical device or implant,
such as a biomimetic material. In another embodiment, the substrate
is a polymer for coating for an implant or medical device.
[0067] The methods further comprise operably linking one or more
peptides to the surface of the substrate. In one aspect of the
methods, the proteins and peptides of the present invention are
grafted to the surface of a substrate. Grafting of the proteins of
peptides can be accomplished through any process or method known in
the art, including, for example, incorporating the peptides into
the substrate material, or crosslinking, photocrosslinking,
bioconjugation, and polymerization. In an exemplary embodiment,
proteins or peptides may be grafted to the surface of a PEG
hydrogel by conjugation of the protein or peptide to
acryloyl-PEG-SVA to produced PEG-peptides, which may then be
incorporated into the surface of the PEG hydrogel through
polymerization of the PEG. In another embodiment, the proteins or
peptides are incorporated into the substrate material, such as by
incorporation into a nanofibrous matrix or multilayer vascular
graft. In another embodiment, the proteins or peptides are
incorporated into a coating that is placed onto a substrate, for
example an implant or medical device.
[0068] Substrates that can be used for methods of the present
invention include polymers as described herein, as well as matrices
and materials, and especially biomimetic materials that form
medical devices or implants. In one aspect, the substrate can be a
coating on a material or matrix, for example a PEG hydrogel coating
on the surface of a polymer. In another aspect, the substrate can
be a biomimetic material, such that no coating is required for
grafting of the proteins or peptides.
[0069] In a further aspect, substrates can include surfaces and
channels for in vitro testing devices, such as microfluidic chips,
and materials that support vascularization such as for use in
engineered tissues.
Medical Device Coatings
[0070] In yet another aspect, the compositions of the present
invention may be applied to medical implants or devices. Medical
implants according to the present invention are preferably vascular
grafts, including stents, shunts, and valves. In one aspect, a
polymer compatible with cellular vascularization is applied to or
coupled to a surface of a medical implant or device that is
comprised of a matrix or material. The application of a polymer to
the matrix or material forming the medical implant or device can
be, for example, through incorporation of the peptides into the
polymer material. Preferred applications include the augmentation
of existing systems and/or polymers for medical device and implant
coatings, which are in long-standing commercial use. These existing
systems and polymers fail frequently, due in many cases to large
extent as a results of lack of endotheliazation. Additional
long-term applications include use in tissue engineered products.
The application of a polymer to the matrix or material forming the
implant or medical device can also be, for example, through
interfacial photopolymerization with the matrix or material.
Matrices or materials for medical devices or implants can include
those intended for tissue engineering, including biomimetic
materials. Specific applications include vascular grafts,
vascularization of synthetic biomaterial, and next generation
stenting.
[0071] To support vasularization and vascular re-endothelialization
and vascular re-endotheliazation, the compositions may comprise
materials currently used for intravascular materials. Materials
used in vivo to support vascularization and re-endotheliazation
include biomimetic materials that are able to maintain adherent
cells on their surfaces under physiological shear stress, support
migration and proliferation of mature endothelial cells from the
ends of the lesion, and provide appropriate ligands for the rolling
and eventual firm adhesion of endothelial colony forming cells
(ECFCs). In a further aspect, therefore, the medical implants of
the present invention may further comprise two or more peptides and
combinations of peptides as described herein. Inclusion of the
peptides significantly increase the capture rate of ECFCs under
shear. The combination of peptides includes an ECFC slowing peptide
and an ECFC capturing peptide. The peptides may be one or more of
CRRETAWAC (SEQ ID NO:1), REDV (SEQ ID NO:2), RGDS (SEQ ID NO:3),
YlGSRG, P11 (SEQ ID NO: 11), and/or PRb (SEQ ID NO: 8). In a
preferred embodiment, the peptides are directly incorporated into a
polymer, which is in turn applied to or coupled to the matrix or
material forming the medical device or implant. Alternatively, the
peptides can be grafted to the matrix or material forming the
medical device or implant.
[0072] In one aspect, the matrix or material used for the medical
device or implant comprises a nanofibrous matrix comprising two
different peptide amphipiles (PAs), which can include one or more
of the peptide described herein, such as REDV (SEQ ID NO:2). PBr
(SEQ ID NO: 8), and/or CRRETAWAC (SEQ ID NO:1). In an alternative
embodiment, a multilayer vascular graft may include a one or more
of the described peptides conjugated into a poly(ethylene glycol)
(PEG) hydrogel to generate bioactive hydrogels that bind to
endothelial cells (ECs). The peptide-incorporating hydrogel may be
reinforced with an electrospun polyurethane mesh to achieve
suitable biomechanical properties. A functional small-caliber
vascular graft must have suture retention strength sufficient for
immediate implantation as well as requisite long-term burst
strength. In addition, graft compliance is not only important in
matching the physical and bulk mechanical characteristics of native
arteries; it has a large effect on preventing reocclusion of
grafts. As mesh thickness is increased, the maximum force that can
be sustained, or suture retention strength in this case, increases.
Mesh thickness has a similar effect on burst pressure of vascular
grafts.
[0073] In a preferred embodiment, electrospinning may be utilized
for fabricating the segmented polyurethane mesh sleeve because this
process produces fibrous, porous scaffolds with mechanical
properties which can be broadly tailored via modification of
electrospinning parameters. The ability to tune the biomechanical
properties of electrospun segmented polyurethanes to improve
matching to those of native vasculature is important in preventing
intimal hyperplasia and thrombosis-induced failure in small
diameter grafts.
Vascularization
[0074] In another aspect, the present invention involves
vascularization of engineered tissues and biomimetic materials. The
compositions of the present invention can be applied to implants
and medical devices, which are then exposed to endothelial cells.
The implants and or medical devices are then incubated with the
endothelial cells under conditions that permit blood vessel
formation. Such conditions may occur in vitro, through cell and
tissue culture, or in vivo, through implantation.
In Vitro Applications
[0075] In a further aspect the invention involves in vitro
applications of the described compositions and methods. In one
aspect, the described peptides can be integrated onto microfluidic
chips, which can be used for drug testing devices. Incorporation of
the peptides onto such microfluidic chips can be performed as
described elsewhere for other substrates. In addition, it is
contemplated that the integration can be through processes such as
protein "printing" and 3D printing. In a further aspect, the chips
can the undergo endotheliazation, to produce chips operably linked
to endothelial cells to allow the devices to test drug
response.
Cell Capture and Diagnosis
[0076] In another aspect the compositions and methods of the
invention can be used for cell capture and diagnosis techniques. In
one aspect, the proteins and peptides and be used for targeted
delivery to ECFCs, for example for delivery of cancer therapeutics.
The proteins or peptides can be operably linked to a drug-delivery
system, such as a particle, nanosphere, or liposome, allowing for
specific targeting of endothelial cells, including ECFCs. In
another aspect, the compositions can be applied to methods of
sorting or separating endothelial cells from a mixed population of
cells, for example through magnetic affinity cell sorting (MACS).
The proteins or peptides can be operably linked to magnetic
nanoparticles to preferentially attach to the endothelial cells
expressing the molecule to which the proteins or peptides bind. In
another aspect, the described compositions can be used for
diagnostics, for example through identification of specific cells
that bind the proteins or peptides as described. The proteins or
peptides can be conjucated to a marker or indicator, such as
streptavidin or a fluorophore, which allows for specific detection.
Cells to which the conjugated proteins or peptides bind can then be
readily detected using known techniques.
EXAMPLES
Example 1: Peptide-Grafted Hydrogels to Capture Endothelial
Progenitor Cells Under Shear for Endothelialization
[0077] In this study, late outgrowth endothelial colony forming
cells (ECFCs), a type of EPCs, were investigated based on their
advantages for use in endothelialization; ECFCs can be isolated
from adult blood, they proliferate rapidly, and they can become
mature ECs. Poly(ethylene glycol) diacrylate (PEG-DA) was chosen as
the base material to test ECFC dynamic adhesion; PEG-DA is able to
resist protein adsorption and therefore served as a "blank slate"
for testing adhesion ligands. Peptides, including RGDS (SEQ ID
NO:3), REDV (SEQ ID NO:2), YIGSRG (SEQ ID NO:4), CRRETAWAC (SEQ ID
NO:1), P11 (SEQ ID NO: 11), PR_b (SEQ ID NO: 8), and RGES (SEQ ID
NO:5), were grafted on the surface of the PEG-DA hydrogels.
Interactions between ECFCs and the peptides were assessed in two
ways: dynamic adhesion and maintenance of adhesion under shear.
Through observation and quantification of ECFC rolling and
retention on peptide grafted hydrogels under shear, dynamic and
static adhesion between ECFCs and peptides was evaluated.
Methods
[0078] Umbilical cord blood ECFCs were used in this study. To
create the peptide-grafted PEG surfaces, PEG-DA was first
photopolymerized to form a hydrogel base. Peptides were conjugated
to acryloyl-PEG-SVA to produced the PEG-peptides, and 0.7
.mu.mol/mL of each PEG-peptide was grafted onto the surface of the
PEG hydrogel base. Shear experiments were performed to examine ECFC
rolling and adhesion on the hydrogel surfaces. Using a Glycotech
parallel plate flow chamber, the ECFC cell suspension was sheared
over the hydrogels at shear rates of 20 s.sup.-1, 40 s.sup.-1, 80
s.sup.-1, and 120 s.sup.-1. Cell rolling events were recorded at 70
fps using a high speed camera. Cell tracking was performed using
ImageJ and Matlab to determine rolling velocities. Finally ECFC
retention on the RGDSQ ID NO:3)-grafted PEG hydrogels was
quantified under superphysiological shear stress.
PEG Peptide Conjugation
[0079] Peptides, either synthesized or purchased (American
Peptide), were first conjugated to acryloyl-PEG-succinimidyl
valerate (acryloyl-PEG-SVA, 3400Da; Laysan Bio) and then grafted
onto PEGDA hydrogels. Peptides, including RGDS (SEQ ID NO:3), RGES
(SEQ ID NO:5), RGDSHHHHHHG (SEQ ID NO:14), YIGSRG (SEQ ID NO:4),
and REDV (SEQ ID NO:2), were first dissolved in sodium bicarbonate
buffer (0.04 mg/.mu.L, pH 8.5). Acryloyl-PEG-SVA was then dissolved
in 1 mL of 50 mm sodium bicarbonate buffer (pH 8.5) for every 10 mg
of peptide to be reacted. The final peptide:acryloyl-PEG-SVA molar
ratio was 1.2:1. The peptide solution was immediately added
dropwise to the acryloyl-PEG-SVA solution with mild vortexing. The
mixture was allowed to react for 4 hrs at room temperature in the
dark with constant mixing. The resulting acryloyl-PEG-peptide was
then dialyzed (molecular weight cutoff 500-1,000, Spectrum Labs)
and lyophilized for storage under argon at -80.degree. C. in a
brown glass vial.
PEG Peptide Conjugation and Confirmation Through Mass
Spectrometry
[0080] Peptide synthesis was confirmed by mass spectrometry. A
small amount of peptide (<1 mg) was dissolved in 10%
acetonitrile and 0.1% formic acid injection solution. Result mass
was obtained using Q-TOF Premier (Waters, Milford, Mass.) and shown
in Appendix A.
Hydrogel Grafting With Peptides
[0081] To form the PEG-peptide grafting solution, the
acryloyl-PEG-peptide was first dissolved in sterile PBS at the
desired concentration (ranging from 0.7 mM to 5.6 mM) and the
photoinitiator was added as described above. After each PEGDA
hydrogel was photocrosslinked, the top plate of the mold was
removed. The hydrogels were rinsed with sterile PBS and briefly
dried with a stream of nitrogen to remove excess PBS on the surface
of the hydrogel. The mold's 0.5 mm PDMS spacers were replaced with
0.7 mm PDMS spacers and a clean glass slide was placed on top. Then
the PEG-peptide grafting solution was injected between the hydrogel
and the new piece of glass and photocrosslinked to the top of the
PEGDA hydrogel under the UV lamp for 7 mM. PEG-peptide grafted
hydrogels were then rinsed and swollen in sterile PBS for
experiments.
Results
[0082] ECFCs were able to form monolayer and maintain cobble stone
morphology on RGDS (SEQ ID NO:3), CRRETAWAC (SEQ ID NO:1) and PR_b
(SEQ ID NO: 8), grafted hydrogels, but not on REDV (SEQ ID NO:2),
YIGSRG (SEQ ID NO:4), and P11 (SEQ ID NO: 11). Rolling velocity of
ECFCs was shown to relate to shear rates and adhesion material
surface. ECFC rolling velocities increased as shear rates increased
up to 120 s.sup.-1 (FIG. 1). All bioactive peptides supported ECFC
rolling as the velocities were well below the cutoff for rolling
velocity. ECFC rolling velocity was found to be significantly lower
on REDV (SEQ ID NO:2)-grafted hydrogels. This suggests that
.alpha..sub.4.beta..sub.1 integrins may be important in ECFC
rolling. ECFC capture events were only observed on hydrogels
grafted with .alpha..sub.5.beta..sub.1 binding peptides, CRRETAWAC
(SEQ ID NO:1) and PRb (SEQ ID NO: 8), at 20 s.sup.-1. Combination
of REDV (SEQ ID NO:2) and CRRETAWAC (SEQ ID NO:1) have
significantly increased the capture rate of ECFC under shear (FIG.
2).
Conclusions
[0083] All tested bioactive peptides supported ECFC rolling,
whereas PEG-DA alone and RGES (SEQ ID NO:5) did not. PEG-DA was
shown to be a viable "blank slate" base material for testing the
ability of grafted ligands, including PEG-peptides, to interact
with rolling ECFCs. Results demonstrated the ability of
.alpha..sub.4.beta..sub.1 integrin-specific peptide REDV (SEQ ID
NO:2) to significantly reduce ECFC rolling velocity as compared to
other tested peptide sequences. Capture on CRRETAWAC (SEQ ID NO:1)
and PRb (SEQ ID NO: 8), suggests that .alpha..sub.5.beta..sub.1,
rather than .alpha..sub.v.beta.3, is the major integrin that is
responsible for ECFC capture under shear. CRRETAWAC (SEQ ID NO:1)
was found to be superior in capturing rolling ECFCs under shear,
and this effect was enhanced by the combination of REDV (SEQ ID
NO:2). Results of this study could be applied in the design of
biomaterials for stent coating and vascular grafts to enhance
endothelialization and improve EPC strength of adhesion under
shear.
Example 2: Identification of Novel Peptides for Slower ECFC Rolling
and Enhanced ECFC Capture Ability
Introduction
[0084] Pepbank is a useful text mining tool that was developed to
identify peptide sequences in MEDLINE abstracts and two public
sources ASPD and UniProt. With this searching tool, peptides that
are relevant to target integrins can be researched. When both
integrins .alpha..sub.5.beta..sub.1 and .alpha..sub.v.beta.3 are
blocked, EPC adhesion was significantly reduced. Therefore, it is
important to identify peptides that bind to these integrins.
Initially, 47 and 88 hits were obtained for
.alpha..sub.5.beta..sub.1 and .alpha..sub.v.beta.3, respectively.
Almost all RGD- and PHSRN (SEQ ID NO:9)-containing peptides were
excluded from the results since RGD is a ubiquitous peptide while
PHSRN (SEQ ID NO:9) is a well-known synergistic peptide that works
together with RGD. For peptides that bind
.alpha..sub.5.beta..sub.1, VILVLF (A5-1; SEQ ID NO:21), and
-(C16)2-Glu-C2-KSSPHSRNSGSGSGSGSGRGDSP (SEQ ID NO:8) (PR_b) were
selected for further testing. HSDVHK (SEQ ID NO:11) (P11) and
NCKHQCTCIDGAVGCIPLCP (SEQ ID NO:12) (V2) were selected for
.alpha..sub.v.beta.3 testing.
Materials and Methods
Peptide Synthesis
[0085] VILVLFG (SEQ ID NO:15) was synthesized using standard
procedures. Mass spectrometry was used to confirm the
synthesis.
[0086] Some alterations in the sequence of the peptide and extra
reactions were involved in the testing of CRRETAWAC (SEQ ID NO:1),
compared to work previously conducted. In prior studies, the
peptide sequence GSSS (SEQ ID NO:16) was added at the N-terminus of
CRRETAWAC (SEQ ID NO:1) to extend the peptide further away from the
substrate interface. In this project, all peptides were coupled to
the acryloyl-PEG that serves as the spacer arm, so GSSS may not be
necessary. Therefore, the original peptide CRRETAWAC (SEQ ID NO:1)
was synthesized for testing in the present work, instead of
GSSSCRRETAWAC (SEQ ID NO:7). In order to create the disulfide
bridge to form the cyclic CRRETAWAC (SEQ ID NO:1) peptide,
Fmoc-Sacetaminomethyl-L-cysteine (Fmoc-Cys(Acm)) was used during
the peptide synthesis because the Acm protection group is stable
during TFA cleavage. The disulfide bridge can be formed subsequent
to cleavage with iodine oxidation as suggested by aapptec. The
peptide was dissolved in 2 mg/mL of 50% acetic acid. Then the
peptide solution was added into 50 mL of 0.1 M iodine solution in
acetic acid. The mixture was then stirred until the yellow color
persists. Aqueous ascorbic acid was added drop-wise to quench the
excess iodine until the mixture is colorless. The mixture was
placed in rotavap to concentrate by evaporation to approximately
one third of the original volume. Mass spectrometry was used to
confirm the production of the peptide and the cyclic CRRETAWACG
(SEQ ID NO:17) should have a molecular weight of 1150 g/mol.
However, the cyclized CRRETAWACG (SEQ ID NO:17) (1150 g/mol) was
only the minor product whereas the major products were shown to be
1221 g/mol and 1292 g/mol which correspond to the peptide with a
single and double Acm protection groups present. After extra iodine
oxidation procedures were followed, similar results were obtained.
Therefore, the peptide CRRETAWAC (SEQ ID NO:1) (instead of
CRRETAWACG (SEQ ID NO:17)) was purchased from American Peptide with
>95% purity. Formation of disulfide bonds between the cysteine
residues was completed by manufacturer as requested.
[0087] Regarding to the original PRb peptide, the C16 hydrophobic
tail, glutamic acid tail connector, and the -C2-tail spacer will be
discarded to reduce peptide synthesis complexity. As a result,
PHSRNSGSGSGSGSGRGDSG (SEQ ID NO:18) (PRb) was synthesized under
standard procedures. Mass spectrometry was used to confirm the
synthesis.
[0088] HSDVHKG (SEQ ID NO:19) was synthesized under standard
procedures. Mass spectrometry was used to confirm the
synthesis.
[0089] NCKHQCTCIDGAVGCIPLCPG (SEQ ID NO:20) (V2) was synthesized
under standard procedures, which in this sequence identifier
includes the G residue from which the peptide was built. Mass
spectrometry was used to confirm the synthesis. However, the
peptide was not readily to be dissolved in aqueous injection
solution for mass spectrometry analysis. Furthermore, many
unexpected by-products were present. Therefore, the V2 peptide was
excluded in this study due to the inability to accurately
synthesize the peptide.
PEG-Peptide Conjugation and Confirmation Through Mass
Spectrometry
[0090] CRRETAWAC (SEQ ID NO:1), PRb (SEQ ID NO:18), and HSDVHKG
(SEQ ID NO:19) were conjugated to PEG as described in Example 1.
Mass spectrometry was used to confirm the conjugation. While
attempting with the PEG-peptide conjugation under organic solvents
as suggested by the existing art, A5-1 peptide precipitated
immediately after the addition of DIPEA, which acts as a base
catalyst. Therefore, A5-1 was excluded in this study due to the
inability to conjugate to acryloyl-PEG for grafting.
Results and Discussion
Adhesion of ECFCs on Novel Peptide-Grafted PEG Hydrogels
[0091] Adhesion of ECFCs on CRRETAWAC (SEQ ID NO:1)-, PRb (SEQ ID
NO:18)-, P11 (SEQ ID NO: 11)-grafted hydrogels was first accessed.
Both CRRETAWAC (SEQ ID NO:1) and PRb supported ECFC adhesion and
spreading. ECFCs were also able to form a monolayer and maintain
their cobble stone morphology (as shown in FIGS. 6A and 6B).
However, P11 (SEQ ID NO: 11)-grafted hydrogels were only able to
support ECFC adhesion, but not spreading (FIG. 6C). On P11-grafted
hydrogels, ECFCs aggregated to form adherent cell clumps. This
suggests that although P11 (SEQ ID NO: 11) was found to have high
specificity for .alpha..sub.v.beta..sub.3, it may not support
regular cellular activities other than cell adhesion. Without the
ability to support cell spreading and the formation of monolayer,
P11 alone could not support ECFC endothelialization which is
critical for healing damaged and diseased blood vessels.
Similar ECFC Rolling Velocity Exhibited by Novel Peptides Comparing
to RGDS (SEQ ID NO:3)
[0092] ECFC rolling was performed on novel peptide-grafted
hydrogels and the results were compared to RGDS (SEQ ID NO:3), REDV
(SEQ ID NO:2), and YIGSRG (SEQ ID NO:4)-grafted hydrogels (FIG. 7,
Table 2, and FIG. 8). All novel peptides supported ECFC rolling as
the velocities were well below the cutoff for rolling velocity.
Regarding the novel peptide, P11 showed the lowest rolling velocity
whereas CRRETAWAC (SEQ ID NO:1) showed the highest rolling velocity
at all shear rates. Comparing to RGDS (SEQ ID NO:3) and YIGSRG (SEQ
ID NO:4), both P11 (SEQ ID NO: 11) and PRb (SEQ ID NO:18) showed
similar rolling velocities and CRRETAWAC (SEQ ID NO:1) showed
significantly higher rolling velocities at all shear rates. ECFC
rolling velocities of all three novel peptides were significantly
higher than REDV (SEQ ID NO:2) at 40 s.sup.-1, 80 s.sup.-1, and 120
s.sup.-1. Some ECFC capture events were observed on CRRETAWAC (SEQ
ID NO:1) and PRb-grafted hydrogels at 20 s.sup.-1. In summary, the
all tested novel peptides support ECFC rolling.
TABLE-US-00002 TABLE 2 Shear Rate (1/s) Peptide 20 40 80 120
CRRETAWAC 131.6 .+-. 16.3 278.4 .+-. 28.6 550.6 .+-. 64.1 828.4
.+-. 82.7 (SEQ ID NO: 1) P11 (SEQ ID 127.5 .+-. 18.4 251.0 .+-.
41.2 508.0 .+-. 86.5 787.6 .+-. 102.1 NO: 11) PRb (SEQ ID NO: 110.3
.+-. 17.7 235.0 .+-. 36.1 485.7 .+-. 67.5 735.1 .+-. 102.6 18) REDV
(SEQ 78.6 .+-. 15.8 183.3 .+-. 43.4 353.8 .+-. 82.0 564.3 .+-.
177.0 ID NO: 2) RGDS (SEQ 102.2 .+-. 20.1 218.7 .+-. 42.4 455.8
.+-. 86.7 739.7 .+-. 121.9 ID NO: 3) YIGSRG (SEQ 103.2 .+-. 18.9
223.2 .+-. 38.0 487.6 .+-. 72.5 737.0 .+-. 107.9 ID NO: 4)
ECFC Capture on CRRETAWAC (SEQ ID NO:1)- and PRb (SEQ ID
NO:18)-Grafted Hydrogels
[0093] Cell capture events were consistently observed on CRRETAWAC
(SEQ ID NO:1)- and PRb (SEQ ID NO:18)-grafted hydrogels at 20
s.sup.-1. Number of captured cells were counted and normalized to
the total number of cells interacted with each hydrogel surface.
During each of the 2 min recordings, 3.74%.+-.1.0% of ECFCs were
captured on CRRETAWAC (SEQ ID NO:1)-grafted hydrogels and
1.14%.+-.0.2% of ECFCs were captured on PRb-grafted hydrogels FIG.
9. Since both of these novel peptides are specific for the integrin
.alpha..sub.5.beta..sub.1, the result of this study strongly
supports that .alpha..sub.5.beta..sub.1 is responsible for cell
capture. This is in agreement with previous studies that have
suggested that initial capture of EPCs is directly dependent on
integrin .alpha..sub.5.beta..sub.1.
Increased ECFC Capture on REDV (SEQ ID NO:2)/CRRETAWAC (SEQ ID
NO:1)-Grafted Hydrogels
[0094] To further evaluate the potential use of CRRETAWAC (SEQ ID
NO:1) and PRb in the ECFC capture for endothelialization, ECFC
rolling on hydrogels that were grafted with combinations of
CRRETAWAC (SEQ ID NO:1) or PRb (SEQ ID NO:18) with REDV (SEQ ID
NO:2) was assessed. In order to maintain consistent grafting of
peptides, a final concentrations of 0.7 .mu.mol/mL of equal molar
mixtures of acryloyl-PEG-peptides precursors were grafted onto the
surface of PEG hydrogels. Hydrogels grafted with REDV (SEQ ID
NO:2)/CRRETAWAC (SEQ ID NO:1) combination (0.35 .mu.mol/mL of REDV
(SEQ ID NO:2)/0.35 .mu.mol/mL of CRRETAWAC (SEQ ID NO:1)) had
significantly increased ECFC capture of 13.4%.+-.2.3% as compared
to CRRETAWAC (SEQ ID NO:1) alone (FIG. 9). This increased ECFC
capture may be due to the contribution from REDV (SEQ ID NO:2)
which has shown that it significantly reduces the rolling velocity.
This theory was supported by the observation of lower rolling
velocity on REDV (SEQ ID NO:2)/CRRETAWAC (SEQ ID NO:1)-grafted
hydrogels (FIG. 10), although no significant difference was found
between the rolling velocities on CRRETAWAC (SEQ ID NO:1) and REDV
(SEQ ID NO:2)/CRRETAWAC (SEQ ID NO:1) at all tested shear
rates.
No ECFC Capture on REDV (SEQ ID NO:2)/PRb-Grafted Hydrogels
[0095] As REDV (SEQ ID NO:2) has a significantly positive effect on
enhancing the ability of CRRETAWAC (SEQ ID NO:1) to support ECFC
capture, it has a negative effect on PRb's (SEQ ID NO:18) ability
to support ECFC capture (FIG. 9). The implementation of REDV (SEQ
ID NO:2) in combination with PRb has completely eliminated PRb's
capability for ECFC capture (from 1.14%.+-.0.2% to 0%). This
unforeseen result was probably due to the significant difference in
length of the two peptides. Unlike CRRETAWAC (SEQ ID NO:1), PRb has
no Cys that forms internal disulfide bridge, so it is considered as
a linear peptide. While REDV (SEQ ID NO:2) has four amino acids,
PRb has 20 amino acids, which is five times longer than REDV (SEQ
ID NO:2). The much longer PRb may have shielded the shorter REDV
(SEQ ID NO:2) which restricted the access of integrin
.alpha..sub.4.beta..sub.1 on ECFCs to REDV (SEQ ID NO:2). This is
also supported by the increase in ECFC rolling velocity on REDV
(SEQ ID NO:2)/PRb-grafted hydrogels compared to PRb alone as shown
in FIG. 10 and FIG. 11.
Conclusions
[0096] Novel peptides were selected to evaluate their capability in
slowing and capturing EPCs. Pepbank was used to identify peptides
that have high affinity and/or selectivity for the integrins
.alpha..sub.5.beta..sub.1 and .alpha..sub.v.beta..sub.3. Although
these peptides have shown great promise in cell adhesion, their
performance in cell rolling and capture under shear is yet to be
evaluated. Although five peptides were identified, three were
assessed due to the success in synthesis and conjugation to
acryloyl-PEG. Assessment of CRRETAWAC (SEQ ID NO:1) and PRb (SEQ ID
NO: 8 or 18) showed that .alpha..sub.5.beta..sub.1 is the major
integrin that is responsible for ECFC capture under shear.
CRRETAWAC (SEQ ID NO:1) was found to be superior in capturing
rolling ECFCs under shear, and this effect was enhanced by the
implementation of REDV (SEQ ID NO:2). PRb also has potential in
capturing ECFCs, although its capturing ability was not observed
when implementing REDV (SEQ ID NO:2). This illustrates the
importance of the peptide length in biomaterial design.
Example 3: ECFC Rolling on Hydrogels
[0097] To identify whether a cell is rolling, the upper velocity
cutoff was determined by flowing ECFCs across the surface of
control PEG hydrogels at different shear rates. Using video
recordings, ECFC rotation on the focal plane of PEG hydrogels was
observed. As shown in FIG. 12, cell tracking showed that ECFCs'
average velocity on PEG hydrogels fell between estimated fluid flow
velocity at a distance of approximately one cell diameter (i.e. 15
.mu.m, the average diameter of ECFCs in suspension) above the
hydrogel surface and the estimated fluid flow velocity at a
distance of approximately one cell radius (i.e. 7.5 .mu.m) above
the hydrogel surface. Therefore, based on these experiments, 40% of
average fluid flow velocity was used as the cutoff velocity for
cell rolling as is shown in FIG. 12.
[0098] ECFC rolling on RGDS (SEQ ID NO:3)-grafted hydrogels was
performed and compared to both PEG hydrogels and RGES (SEQ ID
NO:5)-grafted hydrogels. RGDS (SEQ ID NO:3) is a ubiquitous cell
adhesion peptide, whereas RGES (SEQ ID NO:5) does not support cell
adhesion and served as a control peptide. Velocities of ECFCs on
RGES (SEQ ID NO:5) showed no significant difference as compared to
control PEGDA hydrogels. On the other hand, velocities of ECFCs on
RGDS (SEQ ID NO:3)-grafted hydrogels were significantly decreased
as compared to ECFC velocities on RGES (SEQ ID NO:5)-grafted
hydrogels at all shear rates. As shown in FIG. 13A, the rolling
velocities of ECFCs on RGDS (SEQ ID NO:3)-grafted hydrogels were
103.+-.3 .mu.m/s, 223.+-.14 .mu.m/s, 469.+-.38 .mu.m/s, and
741.+-.28 .mu.m/s at 20 s.sup.-1, 40 s.sup.-1, 80 s.sup.-1, and 120
s.sup.-1, respectively. Representative traces of individual cells'
instantaneous velocities on RGDS (SEQ ID NO:3)-grafted, RGES (SEQ
ID NO:5)-grafted, and control PEGDA surfaces are shown in FIGS. 13B
and C. For each tested surface, a series of extracted frames
following a representative ECFC moving through the flow chamber is
presented in FIG. 14. Some ECFC capture events were observed on
RGDS (SEQ ID NO:3)-grafted hydrogels at 20 s.sup.-1; the total
number of capture events at this shear rate was far less than the
number of rolling cells, however, and, therefore, insufficient for
analysis (data not shown). In summary, the RGDS (SEQ ID
NO:3)-grafted hydrogels supported ECFC rolling at all shear rates
whereas RGES (SEQ ID NO:5)-grafted hydrogels did not support ECFC
rolling. Specific transient binding between ECFCs and RGDS (SEQ ID
NO:3) was demonstrated.
[0099] In addition to PEG-RGDS (SEQ ID NO:3), PEG-YIGSRG (SEQ ID
NO:4) and PEG-REDV (SEQ ID NO:2) grafted hydrogels also supported
ECFC rolling. YIGSRG (SEQ ID NO:4) and REDV (SEQ ID NO:2) are
peptides known to preferentially interact with ECs. Rolling
velocities on YIGSRG (SEQ ID NO:4) were similar to rolling
velocities on RGDS (SEQ ID NO:3); specifically, the ECFC rolling
velocities on PEG-YIGSRG (SEQ ID NO:4) grafted hydrogels were
102.+-.9 .mu.m s.sup.-1, 223.+-.22 .mu.m s.sup.-1, 484.+-.12 .mu.m
s.sup.-1, and 740.+-.10 .mu.m s.sup.-1 at 20 s.sup.-1, 40 s.sup.-1,
80 s.sup.-1, and 120 s.sup.-1, respectively. Surprisingly, PEG-REDV
(SEQ ID NO:2) grafted hydrogels reduced ECFC rolling velocities
substantially more than either RGDS (SEQ ID NO:3)- or YIGSRG (SEQ
ID NO:4)-grafted hydrogels (FIG. 15A). ECFC rolling velocities on
PEG-REDV (SEQ ID NO:2) grafted hydrogels were 79.+-.4 .mu.m
s.sup.-1, 181.+-.6 .mu.m s.sup.-1, 357.+-.6 .mu.m s.sup.-1, and
560.+-.15 .mu.m s.sup.-1 at 20 s.sup.-1, 40 s.sup.-1, 80 s.sup.-1,
and 120 s.sup.-1, respectively (p<0.05 for shear rates at 80
s.sup.-1, and 120 s.sup.-1 compared to RGDS (SEQ ID NO:3) and
YIGSRG (SEQ ID NO:4)). Representative plots of instantaneous
rolling velocities for ECFCs that were tracked across the field of
view at 80 s.sup.-1 and 120 s.sup.-1 are shown in FIGS. 15B and C.
For each tested surface, a series of extracted frames following a
representative ECFC moving through the flow chamber is presented in
FIG. 16. To visualize the distribution of ECFC rolling velocities,
each tracked ECFC is plotted against its rolling velocity in FIG.
17. To allow for better visualization of the data points and for
visual comparison of ECFC velocity distributions between trials,
cells are separated by trial, where each trial was performed using
a separately prepared peptide-grafted hydrogel. At all shear rates,
it can observed that on the REDV (SEQ ID NO:2)-grafted hydrogels a
large number of ECFCs are grouped at lower rolling velocities with
a more sparse distribution of ECFCs at higher rolling velocities,
whereas on the RGDS (SEQ ID NO:3)- and YIGSRG (SEQ ID NO:4)-grafted
hydrogels, the distribution of ECFCs is more even across all the
observed rolling velocities, with more ECFCs tending to have higher
rolling velocities. Upon closer examination, rolling velocities on
REDV (SEQ ID NO:2)-grafted hydrogels at all shear rates were found
to be significantly and positively skewed toward lower velocities
at all shear rates. These results show that multiple peptides are
able to support ECFC rolling, including RGDS (SEQ ID NO:3), YIGSRG
(SEQ ID NO:4), and REDV (SEQ ID NO:2). In addition, RGDS (SEQ ID
NO:3) and YIGSRG (SEQ ID NO:4) supported ECFCs rolling at similar
velocities, whereas REDV (SEQ ID NO:2) slowed ECFC rolling velocity
to a significantly greater extent. This significant decrease
demonstrates that ECFC rolling velocity on the hydrogels depends on
the particular grafted PEG-peptide and suggests that the integrin
bound by REDV (SEQ ID NO:2), .alpha..sub.4.beta..sub.1 may be
important in ECFC rolling.
[0100] The above specification provides a description of various
methods of generating three-dimensional cell cultures or tissues,
compositions of the same, methods of use, treatment and diagnosing.
Since many embodiments can be made without departing from the
spirit and scope of the invention, the invention resides in the
claims.
Sequence CWU 1
1
2119PRTArtificial SequenceSynthesized peptide 1Cys Arg Arg Glu Thr
Ala Trp Ala Cys1 524PRTArtificial SequenceSynthesized peptide 2Arg
Glu Asp Val134PRTArtificial SequenceSynthesized peptide 3Arg Gly
Asp Ser146PRTArtificial SequenceSynthesized peptide 4Tyr Ile Gly
Ser Arg Gly1 554PRTArtificial SequenceSynthesized peptide 5Arg Gly
Glu Ser1627DNAArtificial Sequencesynthesized nucleic acid; N
represents any nucleotide (A, C, G, or T) and K represents either G
or Tmisc_feature(4)..(5)n is a, c, g, or tmisc_feature(6)..(6)k is
g or tmisc_feature(7)..(8)n is a, c, g, or tmisc_feature(9)..(9)k
is g or tmisc_feature(10)..(11)n is a, c, g, or
tmisc_feature(12)..(12)k is g or tmisc_feature(13)..(14)n is a, c,
g, or tmisc_feature(15)..(15)k is g or tmisc_feature(16)..(17)n is
a, c, g, or tmisc_feature(18)..(18)k is g or
tmisc_feature(19)..(20)n is a, c, g, or tmisc_feature(21)..(21)k is
g or tmisc_feature(22)..(23)n is a, c, g, or
tmisc_feature(24)..(24)k is g or t 6tgtnnknnkn nknnknnknn knnktgt
27713PRTArtificial SequenceSynthesized peptide 7Gly Ser Ser Ser Cys
Arg Arg Glu Thr Ala Trp Ala Cys1 5 10824PRTArtificial
SequenceSynthesized peptide; Xaa is 16 carbons-glutamic acid-2
carbonsPEPTIDE(1)..(1)Xaa is 16 carbons-glutamic acid-2 carbons
8Xaa Lys Ser Ser Pro His Ser Arg Asn Ser Gly Ser Gly Ser Gly Ser1 5
10 15Gly Ser Gly Arg Gly Asp Ser Pro 2095PRTArtificial
SequenceSynthesized peptide 9Pro His Ser Arg Asn1
51010PRTArtificial SequenceSynthesized peptide 10Ser Gly Ser Gly
Ser Gly Ser Gly Ser Gly1 5 10116PRTArtificial SequenceSynthesized
peptide 11His Ser Asp Val His Lys1 51220PRTArtificial
SequenceSynthesized peptide 12Asn Cys Lys His Gln Cys Thr Cys Ile
Asp Gly Ala Val Gly Cys Ile1 5 10 15Pro Leu Cys Pro
20136PRTArtificial SequenceSynthesized
peptidemisc_feature(2)..(4)Xaa can be any naturally occurring amino
acid 13His Xaa Xaa Xaa His Lys1 51411PRTArtificial
SequenceSynthesized peptide 14Arg Gly Asp Ser His His His His His
His Gly1 5 10157PRTArtificial SequenceSynthesized peptide 15Val Ile
Leu Val Leu Phe Gly1 5164PRTArtificial SequenceSynthesized peptide
16Gly Ser Ser Ser11710PRTArtificial SequenceSynthesized peptide
17Cys Arg Arg Glu Thr Ala Trp Ala Cys Gly1 5 101820PRTArtificial
SequenceSynthesized peptide 18Pro His Ser Arg Asn Ser Gly Ser Gly
Ser Gly Ser Gly Ser Gly Arg1 5 10 15Gly Asp Ser Gly
20197PRTArtificial SequenceSynthesized peptide 19His Ser Asp Val
His Lys Gly1 52021PRTArtificial SequenceSynthesized peptide 20Asn
Cys Lys His Gln Cys Thr Cys Ile Asp Gly Ala Val Gly Cys Ile1 5 10
15Pro Leu Cys Pro Gly 20216PRTArtificial SequenceSynthesized
peptide 21Val Ile Leu Val Leu Phe1 5
* * * * *