© 1995 American Heart Association, Inc.
Articles |
Basic Fibroblast Growth Factor Is a Signal for the Initiation of Centrosome Redistribution to the Front of Migrating Endothelial Cells at the Edge of an In Vitro Wound
From the Vascular Research Laboratory, Department of Pathology, Banting and Best Diabetes Centre, University of Toronto, The Toronto Hospital Research Institute, Toronto, Canada.
Correspondence to Dr Avrum I. Gotlieb, Vascular Research Laboratory, The Toronto Hospital, 200 Elizabeth St, CCRW 1-857, Toronto, Ontario, Canada M5G 2C4.
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Abstract |
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Abstract Rapid, efficient repair of the endothelium following focal endothelial wounding and denudation is regulated by a complex series of cellular processes. Directed cell migration, an early essential event in repair, is thought to be initiated by centrosome redistribution toward the front of the cell prior to the onset of migration. As such, centrosomal polarity may be an important regulatory event in directed endothelial cell migration. Little is known about the regulation of this process. To study this further, in vitro wounds were created down the middle of confluent porcine aortic endothelial monolayers by mechanical denudation. Conditioned media collected 1 hour after wounding contained basic fibroblast growth factor (bFGF). Antibodies directed against bFGF added to the cultures at the time of wounding significantly inhibited cell migration and transiently inhibited centrosome redistribution. When transcription was transiently inhibited with actinomycin D, present at 1 hour before and for 1 hour after wounding, the cells moved more slowly (5.2±2.8 versus 22.7±5.7 µm/h for control), taking five times longer for the wound to close. Throughout this period, centrosomes did not reorient to the front of the cells. When either recombinant bFGF or conditioned medium collected from control cultures at 1 hour after wounding was added 23 hours after actinomycin D was washed out (at which time RNA synthesis returned to control levels), the centrosomes redistributed to the front of the cells, and cells migrated at a rapid rate (17.2±4.2 µm/h), similar to control. However, the recombinant bFGF or conditioned media had no effect when added immediately after actinomycin D was removed, ie, when RNA synthesis was still inhibited. Thus, bFGF initiates centrosome redistribution by stimulating processes that lead to the transcription of as yet unknown essential gene(s) that are induced immediately following wounding, and this appears to be at least one mechanism by which bFGF enhances aortic endothelial migration and repair at the site of an endothelial wound.
Key Words: centrosome • microtubules • basic fibroblast growth factor • microfilaments • actinomycin D
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Introduction |
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Under physiological conditions an intact endothelial monolayer, which is required for the normal functioning of the blood vessel wall, is maintained by cellular processes that regulate rapid endothelial repair of very small wounds.1 2 3 Larger areas of denudation are also present on the surface of complicated atherosclerotic plaques, in vessels traumatized by atherectomy and angioplasty procedures, and in saphenous vein bypass grafts. Rapid, efficient repair is essential to reestablish a structurally intact endothelium in these vessels, but the regulation of this complex repair is not well understood.
Centrosome redistribution toward the front of endothelial cells is an essential early event that initiates directed cell migration.4 5 6 7 Very small endothelial wounds, ie, fewer than five cells, repair by lamellipodia extrusion of neighboring cells without centrosome redistribution and without cell migration. However, in larger wounds, where migration is required for reendothelialization, centrosomes redistribute to the front of the cell prior to the onset of migration and well after the onset of lamellipodia extrusion at the front of the cell. Little is known about the regulation of centrosome redistribution. Redistribution of centrosomes requires gene transcription at the time of wounding8 ; inhibition of centrosome redistribution results in a dramatic delay in wound repair, and much of the repair occurs primarily through cell proliferation and not migration.9 The signal that induces this transcription and the genes involved are not known.
Soluble factors have been shown to regulate endothelial migration and repair. Basic fibroblast growth factor (bFGF) is a mitogen for endothelial cells10 11 and stimulates their migration.10 12 13 bFGF can also stimulate the production of plasminogen activator and procollagenase,10 14 which aid in the cell's ability to migrate. bFGF-induced cell migration and proliferation can be inhibited by neutralizing antibodies to bFGF,10 indicating that bFGF is required for these functions.
In the present study we tested the hypothesis that the stimulatory effect of bFGF in endothelial wound repair is due to its action as a signal for the induction of unknown genes at the time of wounding that are required for centrosome redistribution.
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Methods |
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Cell Cultures
Endothelial cells were harvested from slaughterhouse porcine aortas by using the collagenase enzyme dispersion method.3 Cultures were grown in medium M199 supplemented with 5% fetal bovine serum (FBS; Hyclone Laboratories Inc), 50 U/mL penicillin, 50 µg/mL streptomycin, and 0.25 µg/mL amphotericin B (M199 and 5% FBS). Cultures were fed every 2 days. Subculturing was carried out on confluent cultures using 0.05% trypsin and 0.02% EDTA. Cells from passages 2 through 4 were used in the wounding experiments. All other tissue culture reagents were obtained from GIBCO Laboratories.
Wound Model
In the double-sided in vitro wound model used,4
endothelial cells were seeded in 60-mm dishes containing 22x40-mm
sterile glass coverslips. Two days after the culture reached
confluency, a 1500-µm wound was made down the middle of the monolayer
by completely removing the cells in this region with a flat-edged
polytetrafluoroethylene-covered spatula. The time of wounding was
designated as time 0 hours. The wound area was marked by small
scratches made with a sterile diamond pencil. The dishes were rinsed
four times over a period of 2 minutes with sterile phosphate-buffered
saline (PBS) containing Ca2+ and
Mg2+ to remove cellular debris. The wound was
examined under a phase-contrast microscope with a x10 phase objective
to ensure that all endothelial cells were removed. The wounded cultures
were then incubated with M199 and 5% FBS and fed every 2 days.
To determine the rate of wound closure, the wounded cultures were examined every 12 or 24 hours under phase optics on an inverted microscope with a x10 objective and a x10 ocular lens equipped with a 1x1-cm net micrometer. The distance between the wound edges was measured at three different points that were identified by scratches placed along the edge of the coverslip at the time of wounding. Percent closure (100x[1-(wound width at a particular time/original wound width)]) was determined. All experiments were performed in triplicate. As the wounds neared closure, each dish was examined individually every 15 minutes. The time of wound closure was defined as the time when the two migrating wound edges came into contact with each other along the entire length of the wound.
Fluorescent Staining
Intact confluent monolayers and wounded cultures were double
stained at various times after wounding to localize microfilaments and
microtubules.4 Rhodamine-labeled phalloidin (Molecular
Probes) was used to localize microfilaments, and microtubules were
detected with anti–
-tubulin monoclonal antibody (Clone DM-1, Sigma
Chemical Co) followed by fluoresceinated donkey anti-mouse
immunoglobulin G (Jackson ImmunoResearch Labs).
Analysis of Centrosome Position
To determine centrosomal location in cells along the wound edge,
the wounds were fixed at various times and stained for tubulin. The
location of the centrosome was determined by using the nucleus and the
wound edge as reference points.4 The centrosome in each
endothelial cell participating in wound repair was classified as being
toward, middle, or away with respect to the nucleus and the front of
the cell in relation to the wound edge or the direction of cell
migration. A total of 200 cells, 100 from each side of the wound, were
analyzed to determine the percentage of cells with centrosomes in each
of the three locations.
Detection of bFGF in the Conditioned Media
Samples of conditioned media were initially collected during the
last hour of incubation, either before wounding or 30 minutes after
wounding, at 1-hour intervals from 1 through 7 hours, and at 16, 19,
24, 48, and 60 hours and were analyzed for the presence of bFGF by an
enzyme-linked immunosorbent assay (ELISA) using the Quantikine bFGF
immunoassay kit (R & D Systems). Time points 0, 1 through 7 hours,
and 19, 24, and 60 hours were repeated four times. Briefly, blank,
recombinant bFGF standards (5 to 320 pg/mL), bovine acidic FGF (Sigma),
and samples of conditioned media (all in duplicate) were added to
individual wells of a 96-well plate that had been precoated with a
monoclonal bFGF antibody. The assay plate was incubated with the
appropriate samples for 2 hours, removed, washed three times, and then
incubated with a horseradish peroxidase–conjugated polyclonal bFGF
antibody for 2 hours. Subsequently, the plate was washed three times
again and then incubated for 20 minutes with tetramethylbenzidine. The
reaction was stopped with a premixed stop solution containing 6N
sulfuric acid and read spectrophotometrically on a Titertek Multiscan
Plus plate reader at a wavelength of 450 nm. The concentration of bFGF
in picograms per milliliter in the conditioned media was determined by
using a standard curve generated with the bFGF standards.
Neutralization Studies Using Anti-bFGF Antibodies
Immediately after wounding, the cultures were washed with PBS
and incubated in media in the presence or absence of 40 µg/mL rabbit
anti-bFGF polyclonal antibody (Biomedical Technologies Inc). As a
control, 100 µg/mL of rabbit immunoglobulin (DAKO Corp) was used. The
cells were fixed 4 or 24 hours after wounding, and the position of the
centrosomes in the cells along the wound edge was determined. The
percentage of inhibition of wound closure at 24 hours after wounding in
the presence of antibody was determined.
bFGF Recovery of Actinomycin D–Treated Cells
We used a dysfunctional repair model in which a transient
inhibition in transcription at the time of wounding reduced the
efficiency of wound repair.8 Briefly, endothelial
monolayers were preincubated for 1 hour at 37°C with 0.25 µg/mL
actinomycin D (Calbiochem-Novabiochem Corp). The monolayer was then
wounded, washed three times with PBS containing
Ca2+/Mg2+ to remove
debris, and incubated at 37°C with actinomycin D for another hour.
Subsequently, the drug was removed by washing the monolayers four times
with PBS containing
Ca2+/Mg2+, and
fresh medium was then added to the cultures. The cultures were fed
every 48 hours for the duration of the experiment. Using
[3H]uridine incorporation studies, it was determined that
following the 2-hour incubation with actinomycin D, RNA synthesis was
reduced to 5% of control levels, returning to normal levels by 24
hours after actinomycin D was washed out.8 To determine if
bFGF was capable of reversing the effects caused by the actinomycin D
treatment, cultures were incubated with recombinant bFGF (Upstate
Biotechnology Inc) or conditioned medium collected from control
cultures 1 or 24 hours after wounding. The bFGF or conditioned medium
was added either immediately after the actinomycin D was removed from
the cultures or 23 hours after the drug was removed. In some cases the
bFGF was preincubated with the neutralizing anti-bFGF antibody for 2
hours at 37°C before being added to the cells.
Statistical Analysis
The times of wound closure and the centrosome positions of the
different treatment groups were compared by a repeated-measure ANOVA.
If a significant difference was seen, then a Newman-Keuls test was
performed to determine which treatments were significantly different
from each other. Statistics were performed by using the
STATVIEW 4.01 program (Abacus Concepts Inc) on a
Macintosh IIsi computer.
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Results |
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Normal Wound Repair
In agreement with previous studies,4 following wounding of the normal endothelial monolayer cells moved at an average rate of 23.4±3.9 µm/h, with the wound closing in 61±2.8 hours (Fig 1
). Within 1 hour after wounding, the centrosomes
redistributed toward the front of the cells (ie, the centrosomes were
between the nucleus and the wound edge), peaking by 3 hours, when 81%
of the centrosomes were facing toward the wound edge. This peak level
was maintained until the wound closed. Within hours after the wound
closed, the centrosomes lost their polarity, becoming completely
randomized by 36 hours after closure (Fig 2A). Following
the initiation of centrosome redistribution after wounding, the dense
peripheral band of actin microfilaments disappeared, and the central
microfilament bundles increased. The dense peripheral band returned 40
hours after wound closure.
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) and neutralizing anti–basic fibroblast growth
factor (bFGF) antibody (
)–treated cultures. The addition of 40
µg/mL of anti-bFGF antibody to the cultures immediately after
wounding significantly reduced the rate of repair compared with
control. The addition of 100 µg/mL rabbit immunoglobulin (
) had
no effect on wound repair. *P<.001 at all time points vs
control.
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P<.05 for centrosomes toward wound vs time 0.
Detection of bFGF Released Into Conditioned Media
The concentration of bFGF found in the conditioned media was
determined by an ELISA using media collected from nonwounded cultures
at various times after wounding. The results were plotted as the
concentration of bFGF (Fig 3). There was no bFGF
detectable in conditioned media from nonwounded confluent cultures. At
30 minutes after wounding, 36.9 pg/mL bFGF was detected in the
conditioned media. This increased by 1 hour (132.6±27.58 pg/mL), was
reduced at 2 hours (32.3±8.52 pg/mL), and was not detectable at 4
hours after wounding (Fig 3). After this time no bFGF was detectable in
the conditioned media, as measured at 5, 6, 7, 16, 19, 24, 48, and 60
hours. There was no detectable bFGF present in the control media
containing M199 and 5% FBS. Acidic FGF (50 ng/mL) showed no
cross-reactivity with the anti-bFGF antibodies used in this ELISA (data
not shown).
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bFGF Neutralization Studies
There was transient inhibition in centrosome redistribution and a
significant reduction in the rate of wound closure when 40 µg/mL
neutralizing anti-bFGF antibody was added to the cultures immediately
after wounding (Fig 1
). At 4 and 24 hours after wounding, the
centrosomes were still randomly associated around the nucleus (Fig 2B).
By 48 hours after wounding, 65.7±3.5% of the centrosomes
redistributed to face toward the wound edge, peaking at about 80% at
96 hours (Fig 2B). Normally it takes only 3 hours for 80% of the
centrosomes to redistribute toward the wound edge (Fig 2A). It took 2.6
times longer for the wound to close due to the addition of the
neutralizing antibody (Fig 1) (61±2.8 hours for control versus
158±4.7 hours for treated cells). Nonspecific rabbit immunoglobulin
(100 µg/mL) had no effect on centrosome redistribution or cell
migration.
bFGF Recovery of Dysfunctional Repair
We used a model of dysfunctional repair in which actinomycin D was
used to transiently inhibit transcription at the time of
wounding.8 Actinomycin D was added 1 hour before and for 1
hour after wounding and then removed by a series of washes. This caused
the rate of wound closure to be significantly reduced. The cells moved
at a peak rate of only 2±2.8 µm/h, with the wound taking 293±7.8
hours to close (Fig 4). During this time, the
centrosomes remained randomly associated around the nucleus (Fig 5A). The dense peripheral band remained prominent for
the first 144 hours after wounding and then began to break down as the
central microfilament bundles increased.
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) or with actinomycin D followed by 5 ng/mL basic
fibroblast growth factor (bFGF) added immediately after the removal of
actinomycin D 1 hour after wounding (
), 5 ng/mL
bFGF that had been preincubated with 40 µg/mL anti-bFGF antibody
added 24 hours after wounding actinomycin D–treated cells (
),
conditioned media collected 1 hour after wounding of control cultures
added 24 hours after wounding actinomycin D–treated cells (
), or
conditioned media collected 24 hours after wounding of control cultures
added 24 hours after wounding actinomycin D–treated cells (
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Preliminary experiments with 0.1 to 10 ng/mL recombinant bFGF showed
that the actinomycin D effect was consistently reversed at 48 hours
when 5 ng/mL bFGF was added 24 hours after wounding; at 0.1 and 0.5
ng/mL, the actinomycin D effect was significantly reversed at 168 and
96 hours, respectively. We used this range of concentrations since the
range of mitogenic, chemokinetic, and chemotactic concentrations used
in the literature was similar.10 11 12 13 When 5 ng/mL
recombinant bFGF was added to the cultures immediately after
actinomycin D was removed, ie, when RNA synthesis was
inhibited,8 the effects of actinomycin D could not be
reversed; the wound still closed slowly (Fig 4), and the centrosomes
did not redistribute (Fig 5B). However, if bFGF was added 23 hours
after actinomycin D was removed, when RNA synthesis was
occurring,8 wound closure occurred two times faster than
in cultures treated with actinomycin D only (145±3.1 versus 293±7.8
hours; Fig 4). This treatment with bFGF also caused a significant
increase in centrosome redistribution, which reached a peak of
68.2±3.4% of the centrosomes facing toward the wound edge by 120
hours after wounding (Fig 5C). The addition of bFGF also caused the
prominent dense peripheral band to break down. Recombinant bFGF (5
ng/mL) that had been preincubated with 40 µg/mL neutralizing
anti-bFGF antibody for 2 hours was not able to reverse the effects
caused by actinomycin D treatment when added to the cultures 23 hours
after actinomycin D was removed (Fig 4). When conditioned medium
collected from control cultures (which contained bFGF released from
injured cells; Fig 3) 1 hour after wounding was added 23 hours after
the actinomycin D was washed out, wound closure occurred 1.7 times
sooner then cultures treated with actinomycin D only (Fig 4). This
treatment also caused the centrosomes to redistribute in a similar
fashion as control cells, with a peak of 57±1.6% of the cells having
their centrosome facing toward the wound edge. In contrast, conditioned
media collected from control cultures (which contained no detectable
bFGF; Fig 3) 24 hours after wounding and added to the cultures 23 hours
after actinomycin D was removed had no effect: there was no centrosome
redistribution, and the wounds closed slowly (Fig 4). Thus, only bFGF
or conditioned media containing bFGF added after RNA synthesis had
resumed could partially reverse the effects of actinomycin D
treatment.
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Discussion |
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Our results demonstrate that bFGF present at the wound site after wounding is an essential signal for the initiation of gene transcription that is required for subsequent centrosome redistribution, an early and crucial step in initiating endothelial cell migration at a wound edge.4 6 7 Neutralizing anti-bFGF antibody disrupts centrosome redistribution and delays repair. Transcription is required for bFGF to have its effect, but the nature of the genes that are transcribed are as yet unknown.
Centrosome redistribution toward the front of the cell is an essential event in directed cell migration in a variety of cell types, including endothelial cells,4 7 15 16 fibroblasts,6 17 and macrophages.18 Although the physiological importance of centrosome polarity in migrating endothelial cells is not well understood, one important suggestion as to the function of centrosome redistribution is that since the Golgi apparatus relocates in conjunction with the centrosome,6 the Golgi is at a strategically favorable position for the directed insertion of new membrane mass into the leading edge via Golgi-derived vesicles.18 19 The relocation of the centrosome is complex and is not observed in all cell types and under all conditions. For example, in several types of motile blood cells, the centrosome may not always be located in the front of the cell.20 21 The position of the centrosomes in migrating fibroblasts is dependent on the nature of the substratum. Centrosomes redistribute to the front of the cells when migrating fibroblasts are on a two-dimensional surface but remain randomly distributed around the nucleus when migrating within a three-dimensional collagen gel system.22 Fibroblasts migrate in vivo as single cells in a three-dimensional gel. However, endothelial cells lining blood vessel lumens are located on a two-dimensional surface. During repair of the injured endothelium, endothelial cells migrate as a sheet of cells on a two-dimensional surface. Thus, the model used in our experiments mimics in vivo wound repair. The redistribution of centrosomes to the front of the cell has also been observed at an in vivo wound edge.23 24
The effects of shear stress on endothelial centrosome location have been recently studied in single endothelial cells in flow chambers. The centrosome position was not completely dependent on the direction of migration25 but rather was the result of lamellipodia extrusion.26 These studies using isolated endothelial cells may reflect angiogenesis more than large-vessel endothelial repair since in the former sheet migration is not a prominent feature as the cells migrate as single cells to form new capillaries. We have reported that disruption of cell-cell contact in large-vessel endothelial cells is associated with loss of centrosomal polarity toward the wound edge in endothelial cells repairing in vivo wounds.24 More work is needed to understand the influence cell-cell contacts have on cell migration and centrosome polarity during endothelial repair.
bFGF is a potent mitogen for a number of cell types of neuroectodermal, endodermal, and mesodermal origins, including endothelial cells.12 In addition to being mitogenic, bFGF is chemotactic27 and capable of inducing the production of proteases such as plasminogen activator and collagenase.10 27 28 29 30 bFGF is synthesized without a signal sequence on its amino terminal and therefore lacks the structural features normally required for protein secretion. Although bFGF lacks the signal sequence, it can be found outside of the cell. The exact mechanism of bFGF release is unknown, but bFGF can be released via disruptions in the cellular membrane.31 32 33 This is presumably how at least some of the bFGF appeared in the culture media in this study following mechanical injury of the endothelial monolayer. Wounding may also liberate bFGF stored in the extracellular matrix.34 35
bFGF is capable of stimulating reendothelialization in vivo.36 After balloon catheter denudation of the rat carotid artery, regrowth of the endothelium ceases after 6 weeks, leaving a large area of denudation. This cessation of reendothelialization is overcome by the systemic administration of bFGF.36 In vitro studies have shown that bFGF is a potent mitogen for endothelial cells28 as well as stimulating their migration.10 37 Both bFGF-induced cell migration and proliferation can be blocked by the addition of neutralizing antibodies to bFGF.10 Our study has confirmed these findings on reendothelialization and has shown that bFGF may induce its effect on migration, at least in part, by stimulating centrosome redistribution to the front of the cell.
In summary, the location of the centrosome in endothelial cells migrating in a polarized fashion has been considered to be essential for establishing and maintaining cell polarity during directed cell migration. Since we first described centrosome redistribution5 both our group and several others have studied the process of centrosome redistribution in migrating endothelial4 7 8 9 16 26 and other cell types.6 17 18 19 20 21 22 Our previous findings have shown that centrosome redistribution occurs before migration, so it is not an epiphenomenon of migrating cells. Neither is it an artifact of cell culture, as we and others have shown this to occur in vivo.23 24 We have now embarked on studies to identify regulators of centrosome distribution and to understand how they work. Our data linking bFGF to centrosome redistribution are important because they provide the first evidence that bFGF may be linked to cytoskeletal processes that are essential in the regulation of directed cell migration. This finding has important implications for both an understanding of the basic science of directed cell movement and the design of potential therapeutic interventions in reendothelialization after vessel injury such as occurs in atherosclerosis, angioplasty, and venous bypass grafting. A detailed knowledge of the mechanisms controlling endothelial movement opens the possibility of either pharmacological or molecular interventions such as gene therapy that would facilitate the repair of the injured endothelium.
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Acknowledgments |
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This study was supported by grant MT 6485 from the Medical Research Council of Canada. Dr Ettenson is a recipient of a traineeship from the Heart and Stroke Foundation of Canada.
Received September 19, 1994; accepted January 19, 1995.
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