(Stroke. 2000;31:498.)
© 2000 American Heart Association, Inc.
Original Contributions |
Presented in part at the joint annual meeting of the American Society of Neuroradiology and American Society of Interventional and Therapeutic Neuroradiology, San Diego, Calif, May 2328, 1999.
From the Research Center of the Centre hospitalier de lUniversité de Montreál, Hôpital Notre-Dame, Montreal, Quebec, Canada.
Correspondence to Jean Raymond, MD, Research Center of the Centre hospitalier de lUniversité de Montreál, Hôpital Notre-Dame, 1560 Sherbrooke East, Montreal (Quebec) H2L 4 M1, Canada. E-mail notredame.radiologie{at}ibm.net
| Abstract |
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MethodsBilateral carotid aneurysms were constructed with venous pouches in 50 pigs and embolized intraoperatively with collagen sponges with and without GFs (platelet-derived growth factor-BB [PDGF-BB] 0.15 or 1.5 µg or transforming growth factor-ß1 [TGF-ß1] 60 or 600 ng) in each animal. DNA synthesis, cell proliferation, and collagen secretion assays were performed to assess the in vitro effects of GFs on neointimal cells harvested from the treated aneurysms. 125I-PDGF-BB was used to study in vivo GF release from sponges. The thickness of the neointima at the surface of the sponges was measured 2 weeks after surgery. Since porcine aneurysms tend to heal after collagen sponge embolization, this experiment was repeated in dogs, which have shown a propensity for recurrence with the same technique, with 600 ng TGF-ß1 or platelet extracts.
ResultsPDGF-BB stimulated DNA synthesis and cell proliferation, while TGF-ß1 strongly increased collagen synthesis of neointimal cells in vitro. Clearance of 125I-PDGF-BB from the sponges followed a biphasic curve, with 1.5% of exogenous PDGF-BB remaining at 1 week. The local delivery of PDGF-BB (0.15 or 1.5 µg) and TGF-ß1 (600 ng) significantly increased neointimal thickness at the neck of porcine aneurysms, while 60 ng of TGF-ß1 had no demonstrable effect. TGF-ß1 (600 ng) or platelet extracts had no influence on canine aneurysms.
ConclusionsPDGF-BB and TGF-ß1 can stimulate neointimal cells in vitro and neointima formation in vivo, but TGF-ß1 and platelet extracts do not compensate for deficient thrombosis in canine aneurysms. Effects on the long-term results of embolization remain speculative.
Key Words: cerebral aneurysm growth factors pathology
| Introduction |
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A variety of growth factors (GFs), including platelet-derived growth factor-BB (PDGF-BB) and transforming growth factor-ß1 (TGF-ß1), have been implicated in neointima formation and vascular remodeling phenomena.6 7 8 9 10 11 12 13 14 15 16 17 18 We have shown that the neointima that develops at the neck of treated aneurysms can be significantly thickened 2 weeks after embolization by the local delivery of a porcine platelet extract (PE) rich in PGDF-BB and TGF-ß1.4 The goals of the present work were to better define which single GF could reach this stimulation as well as to explore in vitro the mechanisms involved. We studied the in vitro effects of PDGF-BB and TGF-ß1 on proliferation, thymidine incorporation, and collagen secretion by cells explanted from the neointima that formed at the surface of the embolic agent. Clearance of GFs delivered with collagen sponges used for embolization was also determined in vivo with 125I-labeled PDGF-BB. The local delivery of 2 doses of PDGF-BB and TGF-ß1 was tested in vivo with the collagen sponge model in porcine lateral wall venous pouch aneurysms. We demonstrate that neointimal cells respond to GF stimulation by enhanced DNA synthesis and proliferation with PDGF-BB as well as collagen secretion with TGF-ß1. Both PDGF-BB and TGF-ß1 can increase the thickness of the neointima formed at the neck of treated porcine aneurysms 2 weeks after embolization.
We have previously shown with this model that the neointima formed at the neck of canine aneurysms is very thin and that lesions recur consistently.5 We finally assessed whether PE or TGF-ß1, both capable of thickening the neointima in porcine aneurysms,4 can improve healing of canine aneurysms treated by sponge embolization.
| Materials and Methods |
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Pathological Studies
Histopathological and morphometric studies were performed at 1
week in 4 pigs treated on 1 side with 1.5 µg PDGF-BB and at 2 weeks
in all other pigs (n=28). Dogs were studied at 3 weeks (n=7). Carotid
arteries and aneurysms were resected en bloc in fresh
Dulbeccos minimal essential medium (DMEM) (Wisent), and the carotid
arteries were opened longitudinally opposite the aneurysms to
examine the luminal surface of the neointima covering the
neck. A 2-mm axial section of the aneurysms, taken in the
middle of the neck, was sliced from the specimen and used for
neointimal measurement after formalin fixation and paraffin
embedding. The remainder of the aneurysms was used for cell
culture, as described below. Five-micrometer axial sections
were stained with hematoxylin-phloxin-safran and Movats
pentachrome, and the neointimal layer at the neck
of each aneurysm was measured (without knowledge of the nature
of the sponge) in 5 locations, as previously developed and standardized
in our laboratory.3 4 5 Direct measurements were taken with
a computerized image analysis system (Vision 2.0, Clemex
Technologies). Immunohistochemical methods were used to characterize
neointimal cells and cells inside the
sponges.3 4 5
In Vivo Clearance of 125I-PDGF-BB
In 18 pigs, experimental aneurysms were
embolized with collagen sponges containing a mixture of
radioactive PDGF-BB (125I-PDGF-BB 200 ng,
Amersham-Pharmacia) and nonradioactive PDGF-BB (800 ng). After 10
minutes to 1 week, the collagen sponges were removed and
homogenized with PBS, and the remaining radioactivity was
determined by liquid scintillation counting for comparison with control
sponges kept in vitro for identical time periods.
Cell Cultures, DNA Synthesis, Proliferation, and Collagen
Synthesis
Porcine carotid arteries and aneurysms were harvested
under sterile conditions at autopsy and maintained in cold DMEM.
Twenty-four explants (1x1 mm) were prepared from the
neointima at the luminal surface of the sponge
(neointimal cells) and from the carotid media, 2 cm distal
to the aneurysmal neck (carotid smooth muscle cells), according
to a method modified from Ross.3 Cells between
passages 1 and 3 were used for in vitro experiments.
DNA synthesis was measured by 3H-thymidine (Amersham-Pharmacia) incorporation assays exactly as described elsewhere.20
Cell proliferation was measured by WST-1 assay (Roche Diagnostics) according to the manufacturers instructions. Briefly, cells were seeded in 96-well plates at a density of 5x10 3 cells per well in DMEM (100 µL) supplemented with 10% fetal bovine serum (FBS). After 24 hours, the medium was removed, and the cells were treated with appropriate concentrations or combination of GFs or FBS for 48 hours. At the end of the incubation period, 10 µL of WST-1 was added for 2 hours. During this time, viable cells converted WST-1 in a water-soluble formazan dye. Absorbance was then determined at 450 nm with an automated optical density reader.
Collagen synthesis was measured according to the method of Koyano et al.21 Cellular outgrowths were seeded in 24-well dishes at a density of 5x103 cells per well in DMEM supplemented with 10% FBS, followed by 24 hours in DMEM containing 0.5% FBS. After 24 hours, the cells were treated with GFs or FBS for 48 hours and labeled with 10 µCi/mL 3H-proline (Amersham-Pharmacia) supplemented with 50 µg/mL ascorbic acid for the last 24 hours. The labeling medium was removed, and the cell layer was extracted with 500 µL acetic acid (0.5 mol/L) for 24 hours at 4°C on a rocking platform, after brief sonication with a Branson Sonifier (Branson Ultrasonics). The day after, each membrane of a 96-well multiscreen filtration plate (0.65-µm pore size, Millipore) was soaked with 50 µL of 25% trichloroacetic acid (TCA), then 50 µL of cell extract and 50 µL of 50% TCA were added to each well. The plates were incubated for 1 hour at 4°C. The precipitate formed was collected on the filter membranes with a vacuum source, and the filters were washed 3 times with 100 µL of 10% TCA. After drying, the membranes were put into scintillation vials, and the filter-bound radioactivity was quantified by liquid scintillation counting.
Statistical Analysis
All data are expressed as mean±SD. The in vitro effects of GFs
on neointimal and carotid smooth muscle cells were compared
by 1-way ANOVA followed by Students t tests. A value of
P<0.05 was considered significant.
Two-way repeated-measures ANOVA with sides (GF-treated or control) as 1 repeated factor and 4 groups (PDGF-BB 0.15 or 1.5 µg and TGF-ß1 60 or 600 ng) as the other factor was used to compare neointimal thickness at the neck of porcine aneurysms. In the presence of a side-by-group interaction, multiple comparisons were made by the least significant difference procedure.
A linear regression model fitted the 125I-PDGF-BB clearance from sponges. We included dummy variables to adjust separate regressions for the 10-minute to 24-hour period and the 1- to 7-day period and compared the 2 slopes. To stabilize the variance, logarithmic transformation was first applied to the percentage of radioactivity remaining within the aneurysms.
| Results |
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-actin
positive after the first passage.
GFs and DNA Synthesis
DNA synthesis of neointimal and carotid smooth muscle
cells was increased significantly (P<0.005) by 20 ng/mL of
PDGF-BB (Figure 1
). Sixty
nanograms per milliliter of TGF-ß1 decreased
thymidine incorporation in neointimal and carotid smooth
muscle cells (P<0.05), while no significant change was
observed with 6 ng/mL of TGF-ß1. Moreover, 60
ng/mL of TGF-ß1 significantly decreased
PDGF-BBinduced DNA synthesis (P
0.05). Finally, there was
no difference in the basal or FBS-stimulated incorporation of thymidine
between neointimal and carotid smooth muscle cells (data
not shown).
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GFs and Cell Proliferation
Proliferation of neointimal and carotid smooth muscle
cells was increased significantly with 20 ng/mL of PDGF-BB compared
with basal values (Figure 2
). The lower
dose of PDGF (2 ng/mL) had no significant effect on cell proliferation.
Both doses of TGF-ß1 had no significant impact
on cell proliferation, as shown in Figure 2
. However, 60 ng/mL
of TGF-ß1 significantly decreased
(P
0.01) PDGF-BBinduced cell proliferation of both cell
types. Finally, no differences were observed in the basal and
FBS-stimulated proliferation of both neointimal and carotid
smooth muscle cells (data not shown).
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GFs and Collagen Synthesis
A 48-hour incubation with either PDGF-BB or
TGF-ß1 significantly increased collagen
synthesis by neointimal and carotid smooth muscle cells
(Figure 3
).
TGF-ß1 was more potent than PDGF-BB. No
synergistic effect was observed between PDGF-BB and
TGF-ß1. Both cell types responded well to GFs,
but basal collagen synthesis by carotid cells was twice that of
neointimal aneurysmal cells (data not shown).
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In Vivo Studies
Angiographic Results
Porcine Aneurysms
Angiographic results initially or at 2 weeks showed no significant
difference between PDGF-BB or TGF-ß1treated
and control aneurysms. The mean angiographic scores immediately
after embolization were 0.25±0.44 and 0.06±0.25 for PDGF-BB or
TGF-ß1treated and control aneurysms,
while at 2 weeks they were 0.0±0 and 0.36±0.8, respectively. Because
>80% of animals had symmetrical results and >80% of
aneurysms (GF-treated or control) were completely obliterated
immediately after surgery and remained obliterated at 2 weeks, we
stopped performing angiography after the first 20 animals.
Canine Aneurysms
Angiographic results in canine aneurysms are
presented in Table 1
. The mean
angiographic scores immediately after surgery were 1.8±0.8 and
0.7±0.6 for TGF-ß1 or PE-treated and
control aneurysms, while at 2 weeks they were 2.75±0.8 and
2.5±0.7, respectively. The only significant difference
(P=0.003) was between the mean immediate score (1.29±1.3)
and mean score at 3 weeks (2.6±0.9) of all aneurysms,
confirming the tendency of recurrence in canine
aneurysms treated with sponges. TGF-ß1
or PE did not significantly improve the angiographic scores at 3
weeks.
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In Vivo Clearance of125I-PDGF-BB
125I-PDGF-BB clearance from collagen sponges
in vivo followed a biphasic curve (Figure 4
). After logarithmic transformation, the
first rapid clearance phase (between 10 minutes and 24 hours) fitted
the equation 2.86-1.79x(time), while the second slower phase
corresponded to the equation 2.32-0.27x(time). The difference between
the 2 slopes (-1.79 and -0.27) was statistically significant
(P=0.0001).
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GFs and Neointimal Thickness
Porcine Aneurysms
Table 2
presents the data on
mean neointimal thickness at the surface of the sponge in
control and GF-treated aneurysms for each group of animals. The
neointimal layer at the neck of porcine aneurysms
was thick (185 to 439 µm). There was no significant difference
between the control aneurysms of all groups. In addition, no
significant difference was noted between the control and GF-treated
aneurysms in animals treated with 60 ng
TGF-ß1. The neointima in
aneurysms treated with 600 ng TGF-ß1 or
0.15 or 1.5 µg PDGF-BB was significantly thicker than the respective
controls (P<0.005). A significant difference
(P<0.05) in neointimal thickness was observed
between aneurysms treated with 0.15 µg PDGF-BB or 600 ng
TGF-ß1 and those treated with 60 ng
TGF-ß1.
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Canine Aneurysms
The neointimal layer at the surface of sponges in
canine aneurysms was thin or absent (mean thickness, 27 to
61 µm). There was no significant difference in
neointimal thickness between control aneurysms,
aneurysms treated with 600 ng TGF-ß1
(n=3), and aneurysms treated with PE (n=4) (Table 1
).
Pathological Studies
Porcine Aneurysms
One week after surgery, control sponges were covered by a
thick clot containing mainly fibrin, platelets, blood cells, or
inflammatory cells and few
-actinpositive spindle cells. Sponges
treated with 1.5 µg of PDGF-BB were covered with a more advanced
organizing thrombus, infiltrated with and covered by a layer of
-actinpositive spindle cells (Figure 5
).
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At 2 weeks, the collagen sponges were invaded by inflammatory cells and
-actinpositive cells, and a thick neointima was
constantly found at the neck of porcine aneurysms. The
neointima was composed of
-actinpositive and
negative spindle cells and extracellular matrix, including collagen
and macrophages, and was covered by a continuous layer of von
Willebrand factorpositive cells. There was no significant
qualitative difference between histological or
immunohistochemical appearance of the neointima found at
the surface of sponges treated or not treated with GFs (Figure 6
).
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Canine Aneurysms
Collagen sponges were almost intact in canine aneurysms,
even at 3 weeks. The neointimal layer was very thin, was
again composed of
-actinpositive cells (1 to 3 layers) and
collagen, and was covered with von Willebrand factorpositive
cells (Figure 7
).
Histological findings were unchanged by the presence or
absence of TGF-ß1 or PE.
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| Discussion |
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Animal Models and Response to GFs
Venous pouch carotid aneurysm models have been widely used
in experimental studies on healing mechanisms after embolization of
aneurysms.22 23 24 25 26 27 We have used collagen sponges
(Gelfoam) as embolic agents, and we have previously shown that healing
mechanisms are basically similar to those found in aneurysms
embolized with platinum coils.3 Although these 2
embolic agents are certainly different in nature, major species-related
differences are witnessed in animal models, irrespective of the embolic
agent used.3 Gelfoam has been successfully used as a
slow-release vehicle for PE that resulted in thicker
neointima at 2 weeks in treated porcine
aneurysms.4 The main drawback of the porcine model
is a strong tendency to heal after embolization, which does not permit
assessment of the effects of GFs on the incidence of angiographic
recurrences.3 22 This characteristic seems to be
linked to exuberant thrombosis and thick neointimal
formation in this animal, 2 phenomena also found after
angioplasty.31 32 In the present study we chose to aim
at the complete obliteration of aneurysms to minimize
asymmetries in residual blood flow between aneurysms.
Angiography performed in the first 20 animals served to demonstrate
that the surgical technique led to consistent results and that
differences in neointimal thickness found at 2 weeks were
not caused by asymmetries in blood flow. Angiographic cure of porcine
aneurysms at 2 weeks was virtually constant in GF-treated or
control aneurysms. In contrast, recurrences can
reliably be seen after collagen sponge embolization of
aneurysms in dogs, and this was confirmed in our
study.3 5 Deficient healing of canine aneurysms is
associated with thin or absent neointima formation,
probably related to deficient thrombus and consequently the lack of a
provisional matrix, which permits physical support to cell migration
and colonization.5 This deficiency could not be
compensated with TGF-ß1 or PE, which were not
able to increase neointimal thickness or improve the
morphological results at follow-up, at least with the doses and
techniques used in the present study. However, fibrinogen and
vascular smooth muscle cell grafts have been successful in increasing
neointima formation in the same canine model.5
Thus, thrombus formation and deposition of a sufficient provisional
matrix at the surface of the embolic agent may be sine qua non
prerequisites to cellular colonization, and only then can GF
stimulation of neointima formation be demonstrated.
Alternatively, some thrombus-related signal may be essential for GF
stimulation of neointima formation.8
Neointimal Cells and GFs
We have previously reported that cells involved in
aneurysmal healing after embolization can be cultured in vitro
with the collagen sponge model.3 Their in vitro response
to PDGF-BB and TGF-ß1 is reported here for the
first time. The nature and origin of cells involved in
neointima formation after vascular injury or in
atherosclerosis remain a matter of
debate.31 32 33 34 35 36 37 38 39 40 Although circulating stem
cells,38 39
monocytes/macrophages,14 18 35 36 37 and adventitial
myofibroblasts33 35 have been evoked as potential actors
in neointima formation, migration and proliferation of
vascular smooth muscle cells from the adjacent media is a commonly
accepted mechanism.8 9 10 11 12 These cells are thought to
undergo phenotypic modulation to switch from a quiescent contractile
stage to a secretory phenotype, specialized in proliferation
and extracellular matrix secretion.12 17 18 41 42 An
alternative hypothesis is that the arterial media is
composed of multiple cell types and a specific population of cells is
responsible for neointima formation.33 43 44
Cells recovered at the surface of the embolic agent in this model are
-actinpositive smooth musclelike cells that present in vitro
characteristics identical to carotid medial smooth muscle
cells.3 Neointimal cells recovered from
vascular injury models have been reported as cells that proliferate
faster and secrete more extracellular matrix proteins than medial
vascular smooth muscle cells, characteristics that persist for many
passages in vitro.45 46 Aneurysmal
neointimal cells manifested no significant increase in DNA
synthesis or cell proliferation compared with carotid smooth muscle
cells and presented decreased basal collagen synthesis in the
present study. Reports have suggested that neointimal
cells recovered after balloon injury have a blunted response to GFs
compared with normal vascular smooth muscle cells.47 48 49
In the present experiments, aneurysmal
neointimal cells and carotid smooth muscle cells responded
in a fashion similar to exogenous GFs added to the culture media. Thus,
the present study supports the theory that neointimal
cells are related to smooth muscle cells migrating from the
arterial media. An alternative hypothesis is that in vitro
cultures by explantation techniques select a subpopulation of medial
cells that, if proper conditions arise, are also involved in
neointima formation in vivo.34 43 44 This
second hypothesis may be supported by the observation of a thicker
neointima at the surface of sponges seeded with vascular
smooth muscle cells harvested from explants prepared from the femoral
artery.5
The mitogenic effect of PDGF-BB is well known.7 8 9 10 11 12 50 Conversely, the in vitro effects of TGF-ß1 are remarkably diverse and sometimes paradoxical. Depending on cell type, density, and culture conditions, TGF-ß1 may stimulate or inhibit proliferation.51 52 53 54 55 In this study as well as other studies,51 52 53 54 TGF-ß1 slightly inhibited the growth of both neointimal and carotid smooth muscle cells. TGF-ß1 also inhibited the proliferative response to PDGF-BB, a phenomenon also observed with medial or neointimal cells in other models.52 53 54 Both factors could increase collagen secretion by both cell types, but TGF-ß1 was more potent, a phenomenon documented previously.11 15 56
The present in vitro studies were performed to better understand the potential mechanisms of GF stimulation of neointima formation. We demonstrated that cells involved in neointima formation at the neck of aneurysms treated by embolization and cultured in vitro respond to PDGF-BB by enhanced DNA synthesis and to TGF-ß1 and PDGF-BB by increased collagen synthesis. These 2 phenomena could potentially stimulate the formation of a thicker or "stronger" neointima, which it is hoped could decrease the risks of recurrence after endovascular treatment.
GFs and Neointima Formation
PDGF-BB and TGF-ß1 have been shown to
stimulate neointima formation after balloon
injury.6 7 8 9 10 11 12 13 14 15 16 The in vitro response of
neointimal cells to PDGF-BB and
TGF-ß1 are distinct, and yet in vivo effects on
neointima formation cannot be distinguished. The in vivo
actions of GFs are complex and likely part of a cascade
phenomenon.6 7 8 9 10 11 12 13 14 15 16 17 18 57 58 59 60 61 In vitro effects on cells in
artificial culture conditions represent, by necessity, a
simplistic approach to the mechanisms involved. Other hypotheses, which
were not studied here, can also be considered, such as the chemotactic
actions of GFs on inflammatory cells and vascular smooth muscle
cells,10 11 12 60 61 effects on cell
differentiation,62 63 64 thrombogenic properties of
TGF-ß1 in vivo,65 or even
induction of tissue factor expression.66 We have shown in
earlier studies that neointima formation at the neck of
porcine aneurysms could be significantly increased at 2 weeks
by the local delivery of PE rich in TGF-ß1 and
PDGF-BB.4 The present study demonstrates that this
response can be duplicated by the local delivery of 2 different doses
of PDGF-BB and by a high dose of TGF-ß1. The in
vivo response to GFs witnessed in our model seems to be nonspecific,
and one hypothesis is that exogenous GFs encourage recruitment of
inflammatory cells, which are themselves a more persistent source of
GFs and cytokines important to healing phenomena, as proposed
in other experimental studies.65 67
Local GF Delivery
If the thicker neointima observed in our model is a
nonspecific response, perhaps mediated through recruitment of
inflammatory cells, a large quantity of GFs, released within 24 hours,
may be an effective strategy. The study of the in vivo release of
125I-PDGF-BB may support this hypothesis since
added GFs are rapidly liberated from sponges at a stage when few
neointimal cells are detected
histologically (Figure 5
). GF release from
sponges showed at least 2 distinct phases. The initial loss of GFs may
be caused by in vitro preparation and surgical manipulations. The first
rapid clearance phase may be explained by rapid washout with
circulating blood flow at the level of the sponge. The second slower
phase could be related to protection from dilution by clot formation at
the surface of the sponge and to nonspecific binding of PDGF-BB to
collagen or other matrix molecules.65 66 67 68 69 70 Thus, an
alternative theory to account for a thicker neointima at 2
weeks is that a minimal fraction of GFs still bound in situ to matrix
molecules is essential for the stimulation of neointimal
cells witnessed in our in vitro studies.67 70 The ultimate
goal of this work is the design of a modified embolic agent, perhaps a
polymer, that locally releases GFs and stimulates healing after
endovascular treatment of aneurysms.70 Another
strategy to reach this goal is in situ cell-mediated gene therapy of
aneurysms, which could involve overexpression of
GFs.5 It would thus be important to better define which of
these 2 potential mechanisms (cellular recruitment by large initial
dose versus cellular stimulation by slowly released or matrix-bound
GFs) is involved in the therapeutic effect observed in porcine
aneurysms.
Conclusion
Neointimal cells harvested from the neck of
embolized porcine aneurysms respond to PDGF-BB by
enhanced proliferation and to TGF-ß1 and
PDGF-BB by increased collagen secretion. In vivo, both factors can
significantly augment the thickness of the neointima formed
at the neck of embolized porcine aneurysms. The
strategy was not effective in correcting deficient healing observed in
canine aneurysms.
| Acknowledgments |
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ur du Québec and partially supported by Boston
Scientific Ltd. We would like to thank Neil G. Hartman, PhD, France
Berthelet, MD, and Marie-Claude Guertin for expert advice on
isotopical, pathological, and statistical studies, respectively. We
thank Rose-Mai Roy for secretarial assistance and Ovid M. Da Silva for
editorial assistance. Received September 27, 1999; accepted November 2, 1999.
| References |
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-smooth
muscle actin expression in granulation tissue myofibroblasts and in
quiescent and growing cultured fibroblasts. J Cell
Biol. 1993;122:103111.Presented in part at the joint annual meeting of the American Society of Neuroradiology and American Society of Interventional and Therapeutic Neuroradiology, San Diego, Calif, May 2328, 1999.
Department of Neurosurgery, University of California, Davis, Davis, California
| Introduction |
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