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Blood, Vol. 95 No. 2 (January 15), 2000:
pp. 581-585
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Enhanced endothelialization and microvessel formation in
polyester grafts seeded with CD34+ bone marrow cells
Vishwanath Bhattacharya,
Peter A. McSweeney,
Qun Shi,
Benedetto Bruno,
Atsushi Ishida,
Richard Nash,
Rainer F. Storb,
Lester R. Sauvage,
William P. Hammond, and
Moses Hong-De Wu
From The Hope Heart Institute and Providence Seattle Medical Center,
the Fred Hutchinson Cancer Research Center, and the Departments of
Surgery and Medicine, University of Washington School of Medicine,
Seattle, WA.
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Abstract |
The authors have shown accelerated endothelialization
on polyethylene terephthalate (PET) grafts preclotted with autologous bone marrow. Bone marrow cells have a subset of early progenitor cells
that express the CD34 antigen on their surfaces. A recent in vitro
study has shown that CD34+ cells can differentiate into
endothelial cells. The current study was designed to determine whether
CD34+ progenitor cells would enhance vascular graft
healing in a canine model. The authors used composite grafts implanted
in the dog's descending thoracic aorta (DTA) for 4 weeks. The 8-mm × 12-cm composite grafts had a 4-cm PET graft in the center and 4-cm
standard ePTFE grafts at each end. The entire composite was coated with silicone rubber to make it impervious; thus, the PET segment was shielded from perigraft and pannus ingrowth. There were 5 study grafts
and 5 control grafts. On the day before surgery, 120 mL bone marrow was
aspirated, and CD34+ cells were enriched using an
immunomagnetic bead technique, yielding an average of 11.4 ±
5.3 × 106. During surgery, these cells were mixed with
venous blood and seeded onto the PET segment of composite study grafts;
the control grafts were treated with venous blood only.
Hematoxylin and eosin, immunocytochemical, and AgNO3
staining demonstrated significant increases of surface
endothelialization on the seeded grafts (92% ± 3.4% vs 26.6% ± 7.6%; P = .0001) with markedly increased microvessels in
the neointima, graft wall, and external area compared with controls. In
dogs, CD34+ cell seeding enhances vascular graft
endothelialization; this suggests practical therapeutic applications.
(Blood. 2000;95:581-585)
© 2000 by The American Society of Hematology.
| |
Introduction |
Improved healing and function of vascular grafts can be
achieved with a proper seeding technique and an appropriate cell
source. In 1978 Herring et al1 were the first to show the
benefits of seeding vascular grafts with endothelial cells. Zilla et
al2,3 modified the seeding method using mass culture
techniques with homologous serum, and they demonstrated a significantly
improved patency rate in grafts seeded with the cultured endothelial
cells. Several studies have shown that seeding grafts with endothelial cells obtained from external jugular vein, saphenous vein, or adipose
tissue also led to improved graft healing.4,5
We have demonstrated that cells in circulating blood can contribute to
the surface endothelialization of polyethylene terephthalate (PET)
vascular grafts and have named this phenomenon fallout
healing.6 Mature endothelial cells have been shown to
circulate in fresh peripheral blood.7 These cells, detached
from the lining of the cardiovascular tree, may be a source of fallout
healing to endothelialize flow surfaces of impervious PET
grafts.6 On the other hand, polymerase chain reaction
analysis on endothelial cells harvested from the surface of impervious
grafts implanted in irradiated dogs transplanted with bone marrow from
dog leukocyte antigen mismatched, unrelated donors showed a pure donor
genotype,8,9 suggesting that the cellular source of fallout
healing was predominantly from endothelial precursor cells in the bone
marrow. Based on this finding, Fujita et al10 used bone
marrow blood to preclot/seed PET grafts, and they observed an
extensively accelerated endothelialization as early as 1 week after
implantation, but the unwanted side effect of microcalcification was
found at approximately 4 weeks after implantation.
Bone marrow contains pluripotent CD34+ cells, which are
known to give rise to hematopoietic cells. In vitro studies show that they can differentiate into mature endothelial
cells.9,11,12 The purpose of this study was to determine
whether canine bone marrow-derived CD34+ cells have the
in vivo potential to enhance vascular graft endothelialization with an
effect comparable to treatment with bone marrow blood when seeded onto
impervious composite grafts implanted in the descending thoracic aorta
(DTA) of dogs, but avoiding the unwanted early microcalcification.
| |
Materials and methods |
Ten healthy young mongrel dogs were used. This study was approved by
the Animal Care and Use Committee of The Hope Heart Institute. Care and
use of all animals complied with the Guide for the Care and Use of
Laboratory Animals.
CD34+ cell separation
One day before surgery, the dogs were anesthetized and 120 mL bone
marrow was aspirated from the humeri and mixed with heparin at a dose
of 10 IU/mL.13 The marrow was then taken to the Fred Hutchinson Cancer Research Center for separation of CD34+
cells. The marrow cells were Ficoll separated (specific gravity, 1.074)
and washed twice in phosphate-buffered saline (PBS)/2% horse serum.
Cells were incubated at 1 × 108/mL with mouse
antidog monoclonal antibody 1 H614 at 10 µg/mL for 30 minutes at 4°C. They were then washed in PBS/2% horse serum and
were incubated with immunomagnetic rat antimouse IgG1 microbeads (Miltenyi Biotech, Auburn, CA). The cell bead mixture was run through a
magnetic column, and the column was washed several times to remove
nonspecifically bound cells. It was then released from the magnet, and
the positively selected cells were eluted from the
column.15
The separated CD34+ cells were kept in approximately 1 mL
PBS with 2% horse serum overnight at 4°C in a slow rotation
chamber. They were then washed free of proteins, and their viability
was assessed using trypan blue stain. Cells that stained blue were considered nonviable because the dye would have penetrated these cells
and caused the cytoplasm to stain. The number of cells that did not
stain was determined using a counting chamber. An aliquot of these
cells was further analyzed using a fluorescent activated cell sorter
(FACS). Samples were run on a FACScan (Becton-Dickinson, Mountain View,
CA) and list mode data analyzed using WINLIST (Verity Software House, Topsham, ME).
Graft preparation and seeding
A three-component composite, impervious, tandem graft was
constructed by suturing 4-cm long segments of 8-mm diameter standard ePTFE to each end of a 4-cm long, 8-mm diameter crimped knitted PET
graft. This PET segment, including end suture lines, was wrapped in a
silicone rubber sheet, and then the entire tandem composite graft was
externally coated with an adherent, 2-mm-thick silicone rubber layer
(Figure 1A). Thus, the seeding effect could
be strictly evaluated in the central PET segment because the ePTFE
segments at the ends prevented pannus extension from the native aorta, and transmural tissue ingrowth from the perigraft areas was blocked by
the external coating.
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| Fig 1.
Scheme of experimental model.
(A) Design of composite graft. (B) Implantation in the descending
thoracic aorta.
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The central PET segment of the composite graft was seeded using a
modified four-step preclot method. In the first step, 1 mL
CD34+ cell suspension was mixed with 3 mL peripheral venous
blood and pressurized into the lumen and the space between the PET
graft wall and its external silicone rubber sheet wrap, with Fogarty vascular clamps in place at each end of the PET segment. The graft was
rotated for approximately 5 minutes to ensure adequate spread. The
excess mixture of cell suspension and blood from this step was removed
from the lumen and was saved for later use in the fourth step. In the
second and the third steps, 5 mL peripheral venous blood was injected
into the lumen to form a layer of fibrin coagulum in the PET graft wall
and its surface, and a 4F Fogarty balloon catheter was used to remove
excess thrombus from the graft lumen and surface. In the fourth step,
the remaining mixture of blood and CD34+ cells from step 1 was mixed with 5 mL heparinized blood (4 mL blood and 1 mL heparin) to
activate antithrombin III to neutralize the excessive thrombin
remaining on and in the graft surface and wall and to decrease the
graft's thrombogenicity.16-18 This mixture was pressurized
into the lumen. The seeded graft was then kept in a moist sponge at
room temperature for approximately 20 minutes while another team was
preparing to expose and clamp the DTA and make it ready for implantation.
The control graft, which was the same composite used for seeded grafts,
was treated with venous blood without seeding.
Graft implantation
The prepared graft was then placed in the canine DTA (Figure 1B)
with the establishment of a carotid-femoral shunt during aortic cross
clamping. The surgical techniques are described in our previous
publications.6,8 Both the seeded and the control grafts
were implanted using the same technique.
This study included 5 grafts in each of the seeded and control groups,
and all grafts were studied after 4 weeks of implantation.
Specimen evaluation
At the end of the study, after induction of deep anesthesia, 10 000
IU heparin was given intravenously, and the dog was exsanguinated. The
specimen was removed and then gently flushed with
Dulbecco's phosphate-buffered saline solution, opened
longitudinally, rinsed again, pinned flat, and photographed.
Three sets of full-wall thickness longitudinal tissue samples were
taken from each of the ePTFE-PET anastomotic areas and from the center
of the PET graft. Additional samples were taken from areas of interest
in the PET graft. Each set of tissue samples, including 4 adjacent
tissue blocks (approximately 1 cm × 0.5 cm each), was studied as
follows: (1) resin-embedded sections stained with hematoxylin and eosin
for general observations; (2) wax-embedded sections for
immunocytochemical studies, including monoclonal mouse antihuman Factor
VIII/von Willebrand factor (FVIII/vWF) antibody (code M-616; DAKO,
Carpinteria, CA) and canine CD34+ polyclonal antibody for
endothelial cells15 and monoclonal mouse antihuman smooth
muscle  -actin antibody (code M-851; DAKO) for smooth muscle cells;
(3) scanning electron microscopy; and (4) transmission electron microscopy.
The flow surface was then stained with 0.5% silver nitrate and viewed
under a 65× stereomicroscope to assess the endothelial-like cell
coverage (ELCC) score, expressed as the percentage of the flow surface
covered by polygonal endothelial-like cells (ELCs). The most
representative thickness of the graft surface lining, determined by
careful examination of each of the hematoxylin and eosin-stained
sections, was measured with a micrometer. The average value with
standard deviation was then calculated for each group.
| |
Results |
Separation of CD34+ cells
The average number of CD34+ cells isolated in the 5 grafts
in each group was 11.4 4 ± 5.3 × 106 (Table1), and viability was
nearly 100%, as judged by trypan blue exclusion just before seeding.
General observations
All grafts were patent at the time of retrieval, and there was no
sign of infection. In both the seeded and the control groups, composite
grafts were well encapsulated with surrounding pleural tissue, though
it was not firmly attached because of the nature of the silicone rubber
coating. There was a thin rim of dark red blood clot in the space
between the PET and the silicone rubber wrap in test and control
grafts. There was no indication of infarction in the kidneys or other
abdominal organs in any of the 10 dogs studied.
Graft flow surface study
Seeded and control grafts looked similar to the naked eye. However,
under the stereomicroscope, the seeded graft flow surface was seen to
be extensively covered with a thin layer of white or pink glistening
tissue, whereas the control graft surface was covered with a thin layer
of translucent fibrinlike material with some thrombus (Figures
2A, 2B). Silver nitrate staining made it possible to distinguish quantitatively the areas covered with polygonal
ELCs on the flow surface (Figures 2C, 2D). Table
1 shows the ELCC scores for each of the
grafts in the seeded and the control groups; the average score was 92% ± 3.4% on the seeded graft flow surfaces and only 26.6% ± 7.6% on the control grafts (P = .00001; Student t
test). There was no significant correlation between the number of
separated CD34+ cells and the degree of ELCC on the seeded
grafts.
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| Fig 2.
Comparisons of representative seeded and control grafts.
Panels on left show seeded grafts, and panels on right, control grafts.
(A,B) Gross specimens. (C,D) Silver nitrate-stained flow surfaces
(×85). (E,F) Hematoxylin and eosin stain (×145). (G,H) CD34
stain (×580). (I,J) Smooth muscle -actin stain (×560).
(K,L) Scanning electron microscopy (×1000). (M,N) Transmission
electron microscopy (×5400).
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Silver nitrate staining also showed that the surface of the ePTFE
segments in both the seeded and control grafts had no ELC coverage,
except on the pannus zones to the extent of a few millimeters inward
from the anastomotic line. There were rare instances where a small,
isolated ELC patch was found in the middle of the surfaces of the ePTFE
segments of the composite grafts in both control and seeded grafts.
Histologic evaluation
There were no osteocytes, osteoblasts, or microcalcification in the
4-week seeded grafts. Immunocytochemical staining with FVIII/vWF and
CD34 antibody demonstrated that the ELCs delineated by silver nitrate
staining had the nature of endothelial cells.
On the seeded grafts, there was a layer of neointima
approximately 174 ± 53 µm thick consisting of a single layer of
endothelial cells shown to be positive with FVIII/vWF and
CD34+ staining on the surface, and there were varying
amounts of positive -actin staining cells and some inflammatory
cells. Most of the control graft surfaces were covered with a thin
layer of pseudointima approximately 124 ± 94 µm thick. There
were no endothelial cells on the pseudointima, which was largely
composed of a fibrin coagulum with some red cells, macrophages,
neutrophils, giant cells, and occasional -actin positive cells
(Figures 2E-2J). Patches of neointima similar to those covering the
seeded grafts were observed occasionally on the control graft surface.
Scanning and transmission electron microscopic studies showed
a contiguous layer of endothelial cells on the seeded graft surfaces.
Cell morphology varied from a spindle-shape to a fish-scale appearance.
Most of the areas on the control grafts were covered with a film of
proteinlike fibrin material mixed with blood cells (Figures 2K-2N).
In seeded and control groups, the graft wall and the space
between the wall and the external silicone rubber wrap were filled primarily with a fibrinous matrix infiltrated with red blood cells and
some inflammatory cells. The major difference was that in the seeded
grafts, microvessels of varying shapes and sizes, which were positively
delineated by FVIII/vWF and CD34 staining, frequently appeared in the
neointima, the graft wall, and the space between the wall and the
external wrap. Several microvessels showed a tendency to connect with
each other in these areas (Figures 3A-3C). The microvessels in the externally wrapped space appeared to be relatively larger than in the graft wall or intima. In comparison, similar microvessels were less often found in the control grafts.
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| Fig 3.
Antibodies to endothelial cell CD34 stain delineate the
enhanced microvessel formation in seeded grafts.
(A) Inside the neointima and external space of the graft (×75).
(B) Microvessel (arrow) and representative view of continuous linear
staining pattern of a microvessel traversing the graft wall
(arrowheads) (×75). (C) Enlargement of area in B (arrowheads)
(×300).
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| |
Discussion |
CD34+ cells include pluripotent hematopoietic progenitor
cells and are defined by the expression of their surface antigen, which
is a mucin-like cell surface glycoprotein. Angioblasts and hematopoietic stem cells share certain antigenic determinants, including Flk-1, TIE-2, and CD34.19 During embryologic
development, blood islands arise from splanchnopleural mesoderm.
Peripherally located cells are precursors of endothelial cells called
angioblasts, whereas the centrally located cells are hematopoietic
precursor cells. They are thought to come from a common precursor
called the hemangioblast. This is supported by the observation that
hematopoietic cells such as QH 1 and MB 1 in the quail and CD34 and
PECAM-1 in the mouse19,20 also express many molecules in
the endothelium.
Asahara et al11 isolated putative endothelial cell
progenitors from peripheral blood by magnetic bead selection on the
basis of cell surface CD34+ antigen expression that became
spindle-shaped endothelial cells and proliferated for 4 weeks. Rafii et
al9 successfully cultured CD34+ cells and
demonstrated that they can differentiate into endothelial cells in
vitro. Shi et al in 19946 and Kouchi et al in
199821 demonstrated, in different dog models, definite
endothelialization from fallout sources on graft surfaces shielded from
external ingrowth. Subsequent studies by Shi et al8,9
further demonstrated that the fallout endothelial cells are derived
from bone marrow. This led to the study by Fujita et al,10
which showed 80% endothelialization of PET grafts preclotted and seeded with bone marrow blood at 1 week, but some unwanted osteocytes and microcalcification were found in the grafts implanted for 4 weeks. Based on the reports cited above, we developed our experimental design concept of CD34+ cell seeding, hoping
to achieve enhancement of graft endothelialization while avoiding these
undesirable side effects.
The recent availability of the necessary monoclonal antibodies allowed
us to initiate studies on the in vivo functions of canine
CD34+ cells. Monoclonal antibody 1H6, 1 of the 10 monoclonal antibodies that recognize CD34,14 has been used
extensively to isolate canine hematopoietic progenitors for stem cell
transplantation studies (McSweeney PA, unpublished data). Flow
cytometry indicated that a high level of enrichment of
CD34+ cells was achieved and that the viability of the
separated CD34+ cells was nearly 100%, as assessed by
trypan blue exclusion. These cells, seeded in and on the grafts in our
experimental model, had the potential to differentiate once the proper
biologic environment was provided after implantation. Zauli et
al22 showed that the number of progenitor cells, including
CD34+ cells, was increased in cultures stimulated by a
combination of cytokines. Rafii et al9 have further
emphasized the necessity of particular growth factors for the
differentiation of CD34+ cells to endothelial cells.
Studies have demonstrated that platelet-derived growth factor, vascular
endothelial growth factor, and basic fibroblast growth factor can be
released from platelets and other blood cells.22-24 We have
found that the fibrin coagulum in the PET graft interstices and in the
space between the graft wall and the silicone sheet wrap entraps many
erythrocytes, leukocytes, and platelets, potentially providing a rich
source of growth factors to facilitate the maturation of
CD34+ cells into endothelium.
In our experience, the CD34 selection procedure typically depletes
non-CD34+ cells by an order of 3 to 4 logs (Bruno B,
McSweeney PM, unpublished data). The CD34+ cells are
enriched from a starting percentage of approximately 2% in unmodified
bone marrow14 to > 75% after enrichment. The enrichment
of CD34+ cells correlated in our study with an enhancement
of flow surface endothelialization and mural microvessel formation,
strongly suggesting that the CD34+ cells were responsible
for this activity. Although a smaller population of
non-CD34+ cells could be contributing to these processes,
this appears less likely. Analysis of the sample of separated
CD34+ cells showed that contaminating cells were
predominantly monocytes or small lymphocytes that nonspecifically bind
to the microbeads. The latter population cannot be accurately defined
in dogs because of the lack of useful B-cell and natural killer-cell
markers, but we postulate that these are B cells because they express
CD45 and because in human studies B cells are less readily depleted from CD34 cells than other lymphocyte populations. On the other hand,
unlike the bone marrow-seeded grafts,10 we could not find any osteocytes or microcalcification in the 4-week seeded grafts. This
indicated that the growth of the osteocyte and its precursors was
either somewhat inhibited or excluded by the separation procedures.
Our composite graft model was designed so that the only sources of
endothelium were either fallout from the circulating blood or
differentiation of the CD34+ cells trapped in the
interstices of the PET graft and in the space between the PET and outer
silicone rubber sheet wrap after seeding. There have been several
reports of endothelial cell outgrowth from cultured
blood.7,9,11 Lin et al25 recently determined that CD34+ circulating endothelial precursor cells have
greater proliferative potential than the mature endothelial cells that
also circulate in the blood. Our study appears to support this finding,
because the study grafts seeded with CD34+ cells had an
average of 90% endothelial coverage. In contrast, the control grafts
exposed to peripheral blood only, which has been shown to have a mature
endothelial population and a lesser number of precursor cells,25
had endothelial coverage of 26%. However, longer-term studies
are needed to demonstrate further the advantages from CD34+
cell seeding, including long-term patency.
Histologic evidence showed that there were more
microvessels (many containing erythrocytes in the lumen) in the seeded
grafts than in the control grafts. They were located in the neointima, in the graft wall, and in the space between the graft wall and the
external silicone rubber wrap (Figure 3). Clowes et al26 define the role of microvessels in the mechanisms of graft healing, and
their findings suggest that microvessels may be closely associated with, and provide the endothelial cellular source for, graft surface endothelialization. Because our model prevented communication with
existing microvessels in the perigraft tissue, the development of these
new microvessels may be associated with the process of differentiation
of CD34+ cells into endothelial cells.9,25 The
presence of the microvessels also suggests that they can develop in the
absence of existing vessels.
When the experimental techniques become feasible, labeling the isolated
CD34+ cells with a long-lasting marker (which carries
through steps of cell differentiation and division and can be seen in
the mature endothelial cells) will provide definitive evidence that the
enhancement of endothelialization originates and benefits from
transplanted CD34+ cells.
Our observation may have clinical relevance if the CD34 origin of
endothelial cells can be further substantiated by our ongoing studies.
As the process of CD34+ cell separation becomes quicker and
less expensive, it may be possible to perform bone marrow aspiration
and separation during the surgical procedure. The CD34+
cells could then be used to seed small-caliber bypass grafts. This may
be more effective and less time consuming and traumatic than harvesting
arm veins or using autologous endothelial cells, and the unwanted side
effects of bone marrow seeding, such as osteocytes and
microcalcification in the graft wall, could presumably be avoided.
This study demonstrated in the dog that CD34+ cells seeded
into grafts can enhance vascular graft endothelialization and
accompanying microvessel formation. It supports our concept of a bone
marrow origin for these phenomena, and it suggests practical
therapeutic applications from these basic biologic insights.
| |
Acknowledgments |
The authors thank C. R. Bard, Inc, and W. L. Gore and Associates, Inc,
for their donation of graft material. They also thank Dorothy Mungin,
histology technician, Warren Berry, medical photographer, Mary Ann
Sedgwick Harvey, medical editor, and Mary-Ann Nelson, medical illustrator.
| |
Footnotes |
Submitted June 7, 1999; accepted September 16, 1999.
Supported in part by National Institutes of Health grants HL03701,
DK42716, and CA15704.
Reprints: Moses Hong-De Wu, Department of Experimental Surgery,
The Hope Heart Institute, 528 18th Avenue, Seattle, WA
98122.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
Presented to the Western Vascular Society, Whistler, BC, Canada,
September 11, 1998.
| |
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Top
Abstract
Introduction