© 1997 American Heart Association, Inc.
Articles |
Vascular Smooth Muscle Cell Cytotoxicity and Sustained Inhibition of Neointimal Formation by Fibroblast Growth Factor 2–Saporin Fusion Protein
From the Department of Cardiovascular Pathology (A.F., S.J.L., D.H.M., R.V.), Armed Forces Institute of Pathology, Washington, DC, and Prizm Pharmaceuticals (Z.P., J.C., J.M., G.F.P.), San Diego, Calif.
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Abstract Basic fibroblast growth factor (FGF2) is an important mediator of smooth muscle cell (SMC) proliferation following arterial injury that results in neointimal growth. The present study was designed to explore the effects of recombinant FGF2 linked to the ribosome-inactivating protein saporin-6 (rFGF2-SAP) on vascular SMC cytotoxicity and neointimal formation following arterial injury. Cultured rat aortic SMCs were exposed to various concentrations of rFGF2-SAP, FGF2, and saporin-6 (SAP). Incubation with rFGF2-SAP resulted in a decreased number of SMCs beginning at a concentration of 10-9 mol/L. Significant cytotoxicity was observed with as little as a 30-minute exposure of SMCs to rFGF2-SAP. To evaluate the ability of rFGF2-SAP in an in vivo model to reduce neointimal formation, Sprague-Dawley rats underwent carotid artery balloon denudation and received an intravenous bolus of vehicle or 5, 10, 15, or 20 µg/kg rFGF2-SAP on 0, 3, 6, and 9 days after injury. Rats were euthanized at 14 days, and carotid arteries were analyzed by computerized morphometry. The threshold dose for a significant reduction in neointimal area by rFGF2-SAP was 15 µg/kg (47% reduction in neointima). When dosing was extended to include days 16, 19, and 22, the neointima was reduced 33% at 28 days (P=.048). rFGF2-SAP reduced neointima without associated medial thinning or arterial wall dilatation. To determine if rFGF2-SAP directly targets SMCs in vivo, rats underwent carotid injury and received either 15 µg/kg rFGF2-SAP or vehicle on day 0 and at 72 hours, with euthanasia at 78 hours after balloon denudation. Medial SMC number was reduced 46% in the rFGF2-SAP group. Tissue sections from arteries 3 days after balloon injury demonstrated rFGF2-SAP binding to medial SMCs and adventitial cells. Staining for fibroblast growth factor receptor 1 revealed a high level of expression in ballooned arteries 3 and 14 days after injury. Taken together, these results provide a molecular and cellular basis for the observed specificity. Prolonged delivery of rFGF2-SAP can affect the natural history of arterial repair after injury.
Key Words: restenosis • saporin • fibroblast growth factor • neointima
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Introduction |
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The pathophysiology of restenosis involves the complex interaction of cellular elements (platelets, inflammatory cells, endothelial cells, and SMCs) with cytokines and growth factors. Previous experimental studies have suggested that FGF2 is an important mediator of the vascular response following arterial injury. Injured endothelial cells release FGF2.1 2 In the rat carotid artery, an infusion of exogenous FGF2 resulted in a marked increase in medial SMC proliferation measured 41 hours and 2 weeks after balloon arterial injury.3 The mitogenic effect of FGF2 on SMCs appears to require arterial injury, since exogenous FGF2 caused no increase in SMC proliferation when the endothelial surface was intact.3 4 Antibody to FGF2 administered before balloon injury in the rat was associated with an 80% reduction in medial smooth muscle proliferation at 41 hours.5 Although FGF2 has been shown to be an important mediator of medial SMC replication early and late after injury, Olsen et al6 were unable to show a reduction in SMC proliferation when the antibody to FGF2 was infused 4 and 5 days after injury, suggesting that other growth factors mediate the response as well. SMCs that constitute the intimal lesion may remain a target for stimulation by FGF2, since mRNA for FGFR1 has been shown to be present on replicating and quiescent intimal SMCs 5 days and 6 weeks, respectively, after injury.7
The novel strategy of cytotoxins linked to growth factors to inhibit
SMC proliferation has been demonstrated in in vitro studies using a
mutated Pseudomonas exotoxin linked to transforming growth
factor-
.8 9 SAP, an alternative cytotoxin, is a
ribosome-inactivating protein that cleaves adenine from ribose in the
28S RNA of the 60S subunit.10 SAP enzymatically inhibits
protein synthesis by preventing binding of elongation factor 2 to the
60S subunit, resulting in cell death.11 SAP is nontoxic to
cells when it remains extracellular, and mammalian cells have no
receptors for the plant protein. When SAP is chemically conjugated to
FGF2 to produce FGF2-SAP and internalized by cells expressing FGF
receptors, FGF2-SAP acts as a mitotoxin and is lethal to cells. The
cytocidal properties of FGF2-SAP have been used to inhibit solid tumor
growth12 and lens reepithelialization.13
Previous short-term studies using the chemical conjugate of FGF2 and
SAP in injured rat carotid arteries demonstrated reduced numbers of
medial SMCs and inhibition of intimal growth,3 14 but
optimal concentrations, optimal duration of dosing, and the duration of
the inhibitory response were not established. Furthermore, a
recombinant fusion protein, rFGF2-SAP (a homogenous single-chain
monomer compared with the more heterogeneous and variable chemical
conjugate), has been developed15 but has not been
evaluated in a balloon injury experimental model.
The objectives of the present study were (1) to further investigate the kinetics of cytotoxicity using the rFGF2-SAP fusion protein in cultured rat aortic SMCs, (2) to identify dosing requirements for sustained inhibition of neointimal proliferation in the rat carotid artery balloon injury model, and (3) to identify targets of rFGF2-SAP in the damaged arterial wall.
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Materials and Methods |
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Preparation and Purification of rFGF2-SAP Fusion Protein
rFGF2-SAP was expressed and purified as described by Lappi et al15 with modifications by McDonald et al.16 In brief, the soluble mitotoxin expressed in Escherichia coli was purified to homogeneity from cell lysates via expanded bed absorption chromatography followed by cation-exchange, heparin-affinity, and size-exclusion chromatography. The purified protein was formulated in 50 mmol/L sodium citrate, pH 6, containing 80 mmol/L NaCl and 0.1 mmol/L EDTA. The final materials were shown to be >98% pure by SDS-PAGE, reverse-phase HPLC, and size-exclusion HPLC and were stored at -80°C until used. Endotoxin levels were <0.05 EU/mg protein.
Cytotoxicity of rFGF2-SAP on Rat Aortic SMCs in Culture
Rat aortic SMCs were prepared from adult female Sprague-Dawley
rats (Hazelton, Pa) weighing 200 to 250 g by the explant method as
previously described.17 Briefly, explants were obtained
from the thoracic aorta, the adventitial layer was dissected away with
a scalpel blade, and the endothelium was removed with a cotton swab.
The tissue explants were maintained in medium 199 supplemented with
10% FBS, 4 mmol/L L-glutamine, 100 U/mL penicillin G
sodium, and 100 µg/mL streptomycin sulfate in a humidified atmosphere
of 5% CO2/95% air. The medium was replaced every third
day. SMCs were allowed to grow out from the tissue, which was
subsequently removed after 9 to 11 days. After confluence, the cells
were subcultured using 0.05% trypsin and 0.053 mmol/L EDTA. Cells
were routinely subcultured at a 1:5 ratio and used between passages 4
and 6.
SMCs were plated in 24-well tissue culture plates (10 000 cells per well) containing growth arrest medium (medium 199) containing 1 mmol/mL insulin, 5 mg/mL transferrin, and 0.1% bovine serum albumin. The following day, SMCs were exposed, for 72 hours, to media alone, rFGF2-SAP, SAP, or FGF2 at concentrations of 10-11 to 10-7 mol/L. At the end of the incubation period, SMCs were washed in PBS and trypsinized. Cell number was determined with a Coulter particle counter. Experiments were performed in duplicate and repeated three times.
Effect of Incubation Time on Cytotoxicity
SMCs were plated as described above. The following day, SMCs in
duplicate wells were incubated for 0.5, 1, 4, or 72 hours in rFGF2-SAP
at concentrations of 10-11 to
10-7 mol/L. SMCs in media alone served as a
control. At the end of the incubation period, SMCs were rinsed in PBS,
and wells were filled with medium. Cell number was determined 72 hours
after initial exposure to rFGF2-SAP. For each incubation time, a line
plot was generated that related the percent reduction in SMC number to
the concentration rFGF2-SAP. The ID50 was defined as the
inhibitory dose of rFGF2-SAP needed to reduce the average cell number
of duplicate wells by 50%. This experiment was performed three
times.
The Effect of rFGF2-SAP on Neointimal Formation Following
Arterial Injury
Anesthetized (ketamine/xylazine) male Sprague-Dawley rats (250
to 350 g) underwent left carotid artery endothelial injury by
three passes of an inflated (0.3-mL) 2F Fogarty balloon catheter
introduced via the external carotid artery. Animals (n=51) were
randomized to receive 5, 10, 15, or 20 µg/kg of rFGF2-SAP diluted in
phosphate-buffered saline containing 0.1% bovine serum albumin or an
equal volume of vehicle (phosphate-buffered saline containing 0.1%
bovine serum albumin) in control rats on days 0, 3, 6, and 9 after
balloon denudation. rFGF2-SAP and vehicle were administered
intravenously via the tail vein. Pulse dosing every 3 days was chosen
in an effort to widen the therapeutic window for rFGF2-SAP. Rats were
euthanized 14 days after balloon denudation and perfusion-fixed with
Histochoice fixative (Amresco). Twenty-four hours and 1 hour before
euthanasia, rats received an intraperitoneal injection of BrdU (30
mg/kg). One hour before euthanasia, 0.3 mL Evans blue dye was injected
intravenously to mark the denuded arterial segment.
Carotid arteries were removed and immersion-fixed with Histochoice for 24 hours. The entire carotid artery was dehydrated in a series of graded alcohols, cleared with xylene, sectioned into 2.0-mm segments, embedded in paraffin, cut at 4 µm, and stained with hematoxylin-eosin and Movat pentachrome stains. All arterial segments showed intimal thickening, and segments were magnified and digitized. Computerized morphometry (IP Lab Spectrum software) was performed (with the observer blinded to treatment group) on arterial segments to measure the area within the EEL, IEL, media, and neointima. Proliferating intimal and medial SMCs were identified after incubating the slides with a monoclonal antibody directed against BrdU. The total number intimal and medial cells (BrdU positive and negative) were counted in eight randomly selected oil immersion fields (x400) from the arterial segment that demonstrated the largest neointimal area.
In a set of experiments to establish the duration of mitotoxin effect, rats (n=42) were randomized to treatment with intravenous 15 µg/kg rFGF2-SAP or vehicle on days 0, 3, 6, and 9 after carotid injury, with euthanasia on day 28. Another group of rats received either intravenous 15 µg/kg rFGF2-SAP or vehicle on days 0, 3, 6, 9, 16, 19, and 22, with euthanasia 28 days after carotid artery injury. Perfusion fixation, artery harvest, tissue processing, and analysis of sections were performed as described above.
Assessment of Medial SMC Toxicity
Male Sprague-Dawley rats underwent carotid balloon endothelial
denudation as described above. Animals (n=14) were randomized to
receive 15 µg/kg of rFGF2-SAP or vehicle intravenously or vehicle at
0 and 72 hours with euthanasia at 78 hours. On the day of arterial
injury and daily until euthanasia, rats received an intraperitoneal
injection of BrdU (30 mg/kg). One hour before euthanasia, 0.3 mL Evans
blue dye was injected intravenously. Rats were perfusion-fixed, and
carotid arteries were processed for light microscopy as described
above. The arterial segment demonstrating the greatest degree of medial
injury was selected with the observer blinded to the treatment. The
section was magnified (x400), and all medial SMC nuclei (BrdU positive
and negative) were counted. IEL, EEL, and medial areas were measured by
computerized morphometry. Eight randomly selected high-power (x400)
fields from the adventitia were digitized, and all adventitial cells
(fibroblasts, neutrophils, macrophages, and lymphocytes) within the
field were counted. The area of the adventitia was measured, and
adventitial cell density (cells/mm2) was calculated. All
medial SMC nuclei were counted from an arterial segment from the
central portion of the uninjured right carotid artery.
rFGF2-SAP Binding to Tissue Sections
Direct binding of rFGF2-SAP and SAP was performed on sections
from a separate group of rat arteries (n=4) obtained 3 days after
injury; sections of an uninjured artery from the same animal were used
as controls. An antibody against SAP was raised in goats (Chemicon
International, Inc) and purified using a protein A column. Frozen
sections cut at 4 µm were allowed to dry overnight at room
temperature. Sections were hydrated in PBS and incubated in either 0.2
µg/mL rFGF2-SAP or SAP alone for 45 minutes at 37°C. The sections
were washed in PBS and incubated with 15 µg/mL of goat anti-SAP for
45 minutes at 37°C to detect bound rFGF2-SAP or SAP. The goat
antibody was detected with peroxidase-conjugated donkey anti-goat IgG
(Jackson ImmunoResearch) diluted 1:100. The rFGF2-SAP, SAP, and primary
and secondary antibodies were diluted in PBS containing 0.2% gelatin
from cold water fish skin (Sigma Chemical Co) as a protein carrier.
Diaminobenzidene tablets (SigmaFast, Sigma) were used as the
chromogen.
FGF Receptor Immunohistochemistry
Immunohistochemistry was performed on Histochoice-fixed,
paraffin-embedded, 4-µm-thick sections obtained from tissues
collected on days 3 and 14 after arterial injury (n=4 at each time
point). A rabbit polyclonal antibody to the human FGFR1 was purchased
from Santa Cruz Biotechnology. This antibody was raised against
C-terminal peptide residues 808 to 822, PRHPAQLANGGLKRR. Immunostaining
was performed on the TekMate 2000 automated immunostainer (BioTek
Solutions), as described previously.18 Sections were
placed in sodium citrate buffer and heated in the microwave oven for
antigen retrieval. The primary antibody was diluted in PBS containing
0.2% gelatin from cold water fish skin as a protein carrier. The
primary antibody was detected using the ABC kit provided by BioTek
Solutions. Cognate peptides were used to demonstrate staining
specificity. A second monoclonal FGFR1 antibody, generated against the
extracellular domain of FGFR1, confirmed the specificity of the
staining.
Statistical Analysis
Data are expressed as mean±SEM. Differences between groups were
compared using an unpaired Student's t test. A two-tailed
probability of P<.05 was considered significant.
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Cytotoxicity of rFGF2-SAP on Rat Aortic SMCs
The incubation of SMCs with varying concentrations of rFGF2-SAP for 72 hours resulted in a significant decrease in SMC number that was first detected at a concentration of 10-9 mol/L. At higher concentrations of rFGF2-SAP (10-7 mol/L), 90% SMC cytotoxicity was achieved. SAP alone did not reduce SMC number from a concentration of 10-11 to 10-8 mol/L (Fig 1
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The efficacy of rFGF2-SAP–induced SMC cytotoxicity in vitro was also
dependent on the duration of exposure (Fig 2).
Concentrations of rFGF2-SAP of 
22 nmol/L were effective in producing
50% SMC death within 30 minutes of exposure to the drug, indicating
that a prolonged exposure is not required to kill SMCs. Increasing the
exposure time to rFGF2-SAP (0.5 to 72 hours) markedly reduced the
concentration of rFGF2-SAP required to produce 50% lethal cell injury.
For example, a 72-hour incubation time resulted in an 7-fold
decrease in the ID50 of rFGF2-SAP compared with the
30-minute time period.
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The Effect of rFGF2-SAP on Neointimal Formation and
Cellular Proliferation Following Arterial Injury
Pulse dosing every third day (days 0, 3, 6, and 9) with rFGF2-SAP
(5, 10, 15, or 20 µg/kg) resulted in a significant (47%,
P<.03) reduction in neointimal formation in rats receiving
drug treatment at 15 and 20 µg/kg (Figs 3 and 4). rFGF2-SAP did not affect the areas enclosed by the
EEL or IEL or the area of the media assessed 14 days after injury (Fig 3). Medial repair was complete in carotid arteries 14 days after
balloon injury in rats receiving rFGF2-SAP; only occasional foci of
medial SMC loss were present, and no acute cellular necrosis or
inflammation or aneurysm formation was present. rFGF2-SAP was well
tolerated by rats through 15 µg/kg, with no clinical evidence of
toxicity. There was a 40% mortality (hepatic toxicity) in rats
receiving 20 µg/kg, occurring after a mean of three doses of
rFGF2-SAP was administered. In rats receiving 15 µg/kg rFGF2-SAP, the
BrdU labeling index at day 14 (Fig 5A) in the neointima
was similar to that in control rats (6.7±1.5% versus 5.5±1.4%,
respectively; P=NS). There was a trend toward greater SMC
proliferation in the media (1.8±0.8%) in rFGF2-SAP–treated rats
compared with control rats (0.4±0.2%, P=.076; Fig 5A).
Cell density (cells/mm2) in the neointima (9116±510 versus
8722±712, P=NS) and in the media (3185±180 versus
3098±159, P=NS) was similar in rFGF2-SAP–treated and
vehicle-treated animals, respectively. Since the amount of neointima
was significantly less in rFGF2-SAP–treated rats, the total SMC number
was correspondingly less as well.
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To determine whether the inhibition of neointimal formation observed at
2 weeks was maintained at 4 weeks after surgery, rats were randomized
to treatment with intravenous 15 µg/kg rFGF2-SAP or vehicle on days
0, 3, 6, and 9 (short-term treatment) or days 0, 3, 6, 9, 16, 19, and
22 (long-term treatment) after carotid injury, with euthanasia on day
28. There was no reduction in neointimal area in the rFGF2-SAP group
(0.136±0.006 mm2) versus the control group
(0.126±0.010 mm2) with short-term treatment (Fig 6A). Medial areas were similar in both groups
(0.152±0.007 versus 0.151±0.003 mm2), and there were
no significant differences in the areas enclosed by the IEL
(0.437±0.017 versus 0.454±0.011 mm2) and EEL
(0.589±0.022 versus 0.605±0.013 mm2) in
rFGF2-SAP–treated and vehicle-treated rats, respectively. The
neointimal BrdU labeling index (Fig 5B
) was similar in
rFGF2-SAP–treated rats (2.4±0.7%) compared with control rats
(1.9±0.6%, P=NS). There was no difference in the medial
BrdU labeling index (0.5±0.2% in rFGF2-SAP–treated rats versus
0.9±0.5% in control rats, P=NS; Fig 5B).
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With long-term administration of rFGF2-SAP, there was a significant
(P=.048) reduction in neointimal area in the
rFGF2-SAP–treated rats (0.068±0.012 mm2) compared
with control rats (0.102±0.010 mm2, Fig 6B). There
were no differences in medial area (0.145±0.007 versus
0.148±0.005 mm2) or areas enclosed by the EEL
(0.586±0.031 versus 0.630±0.032 mm2) and IEL
(0.447±0.028 versus 0.483±0.029 mm2) in the
rFGF2-SAP–treated group compared with the vehicle-treated group,
respectively. There were no differences in the BrdU labeling index (Fig 5C) in the neointima (2.1±0.4% versus 1.4±0.5%, P=NS) or
media (0.1±0.1% versus 0.05±0.05%, P=NS) between
rFGF2-SAP–treated and vehicle-treated rats, respectively. Cell density
(cells/mm2) in the intima (8229±695 versus 7094±438,
P=NS) and in the media (3085±178 versus 2719±266,
P=NS) was similar in rFGF2-SAP–treated and vehicle-treated
animals, respectively. These results indicate that there were fewer
neointimal SMCs at 28 days in this dosing regimen of rFGF2-SAP compared
with the control condition. There was no mortality in rats receiving
rFGF2-SAP.
Is the Medial SMC the Target of rFGF2-SAP In Vivo?
To determine if rFGF2-SAP is capable of directly targeting medial
SMCs, rats were subjected to balloon injury, drug administration on
days 0 and 3, and euthanasia 6 hours after the last dose (78 hours).
The mean number of medial SMCs in the most injured arterial segment was
148±25 cells in the rFGF2-SAP–treated rats compared with 274±19
cells in the control rats (46% reduction, P=.002;
Table). Expressed as SMC density per unit medial area,
the mean number of medial SMCs/mm2 in the most injured
arterial segment was 1150±265 cells/mm2 in
rFGF2-SAP–treated rats versus 2065±236 cells/mm2 in
control rats (44% reduction, P=.024). The
rFGF2-SAP–treated arteries also had extensive full medial thickness
SMC necrosis compared with focal necrosis in control arteries (Fig 7). The mean number of BrdU-positive medial SMCs in the
most injured arterial segment was 25±7 in the rFGF2-SAP–treated group
versus 34±8 in the control group (26% reduction, P=.46).
Notably, in the adventitia, the mean number of cells/mm2
was 246±57 in drug-treated rats versus 556±100 in control rats (56%
reduction, P=.02). The mean number of medial SMCs per
arterial segment in the uninjured right carotid arteries was similar in
rFGF2-SAP–treated rats (257±42) compared with vehicle-treated rats
(278±49, P=.41), indicating the specificity of the response
to the injured arterial segment.
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Identification of Binding Sites for rFGF2-SAP
rFGF2-SAP was incubated with tissue sections from ballooned and
uninjured arteries harvested and processed 3 days after injury to
assess rFGF2-SAP binding. Extensive binding was observed in the media
(Fig 8). Interestingly, adventitial cells from injured
vessels also demonstrated considerable binding of rFGF2-SAP. When SAP
alone was incubated with tissue sections, no staining was observed
using anti-SAP antibodies, indicating that rFGF2 conferred binding
specificity to SAP.
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Expression of FGFR1 on Intimal and Medial SMCs After
Arterial Injury
Because FGF2 can bind FGFR1 with high affinity, sections of rat
arteries harvested 3 and 14 days after injury were analyzed for FGFR1
distribution. In uninjured arteries, receptor immunoreactivity was
detected in SMCs localized throughout the media (Fig 9A). Competition with the antigen eliminated staining
(Fig 9B). In arteries collected 3 days after balloon injury and vehicle
treatment, FGFR1 immunoreactivity was found in the densely cellular
adventitia (Fig 9C) as well as in medial SMCs. In rFGF2-SAP–treated
animals at 3 days, no FGFR1 immunoreactivity was detected in the
adventitia, and considerably fewer cells were present in the adventitia
(Fig 9D). Fourteen days after injury, in both vehicle and
rFGF2-SAP–treated arteries, immunoreactive FGFR1 was detected in SMCs
in the neointima and media, but the adventitia was negative for FGFR1
staining and contained markedly fewer cells than at earlier time points
(Fig 9E and 9F). In aggregate, FGFR1 staining was less in the
rFGF2-SAP–treated group because these animals had a smaller neointima
and less cellularity in the adventitia.
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Discussion |
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The findings described in the present study demonstrate that the mitotoxin rFGF2-SAP inhibits the neointimal proliferation after arterial injury and extend the work of Lindner, Casscells, and colleagues.3 14 In the study by Lindner et al,3 there was a 53% reduction in medial SMCs 41 hours after injury in rats treated with 120 µg of FGF2 chemically conjugated to SAP. Casscells et al14 demonstrated a significant reduction in the ratio of the area of the intima to the media at 14 days after a single treatment with 75 µg/kg of FGF2 chemically conjugated to SAP. Initial studies with the recombinant fusion protein rFGF2-SAP have been performed in a canine carotid endarterectomy model19 and in canine arteriovenous conduits.20 Mattar et al19 demonstrated a 46% reduction in post–carotid endarterectomy intimal area at 14 days with continuous local delivery of 2 µg/kg per day rFGF2-SAP. There was a 40% reduction in the intimal area at the arteriovenous graft anastomotic site at 14 days with continuous local delivery of 2.7 µg/kg per day rFGF2-SAP.20 No previous studies have examined the acute as well as chronic biological effects of repeated doses of the rFGF2-SAP.
In the present report, we have characterized the biological response to
the recombinant fusion protein, rFGF2-SAP, as opposed to the chemical
conjugate that was used in previous studies.3 14 SAP alone
does not inhibit SMC growth, supporting the concept that the toxin must
be internalized to elicit a cytotoxic effect. The potency of the in
vitro cytotoxic effect is also proportional to the duration of exposure
of SMCs to rFGF2-SAP. In vivo experiments further demonstrate that
pulse bolus dosing every 3 days with rFGF2-SAP inhibits neointimal
formation following arterial injury. A significant reduction in
neointimal size at 14 days is seen with a dose of 
15 µg/kg
administered on days 0, 3, 6, and 9 after balloon denudation.
Importantly, rFGF2-SAP does not affect arterial size (area within the
IEL and EEL) or medial area, and neither acute inflammation nor SMC
necrosis is seen at 14 and 28 days. Necrosis of SMCs may be
an important early event, since it was observed only at day 3.
Sustained inhibition of neointimal formation 28 days after balloon
injury required subsequent dosing (days 0, 3, 6, and 9 plus days 16,
19, and 22) of rFGF2-SAP, raising the possibility that intimal growth
may ultimately "catch up" to control arteries in the absence of
continued treatment.
The mechanism of rFGF2-SAP's effect on neointimal formation in vivo is
probably via its cytotoxic effect on medial SMCs. Three days after
arterial denudation, the cytotoxic effect of rFGF2-SAP is seen, with a
46% reduction in medial SMC number. At 14 days (with four doses of
rFGF2-SAP) and at 28 days (with seven doses of rFGF2-SAP), intimal SMC
density was similar in drug-treated and control animals. Thus, the
smaller intimal area in rFGF2-SAP–treated animals corresponds to a
reduction in the total number of intimal SMCs and not cell density. The
reason for the trend toward increased medial SMC proliferation that was
seen on day 14 after four doses of rFGF2-SAP is uncertain and may
reflect rebound from the cytotoxic effects of initial drug treatment.
It should be noted, however, that the 14-day time point is 5 days past
the last rFGF2-SAP treatment; thus, the lack of detectable reduction in
SMC proliferation (Fig 5A) may have been obviated by analysis nearer to
the time of dosing, since marked effects were observed at day 3 when
animals were exposed to the drug on days 0 and 3. Further, these
proliferating cells may be targets for cytotoxicity by subsequent doses
(on days 16, 19, and 22) of rFGF2-SAP, resulting in the chronic
inhibition observed at day 28. This finding may explain why further
doses of rFGF2-SAP are required to ultimately suppress neointimal
growth at 28 days.
The results of the present study showing sustained inhibition of neointimal growth with rFGF2-SAP are supportive of an important role of FGF2 beyond the early post–arterial injury period. These data differ from previous work by Lindner and Reidy,5 in which a polyclonal antibody directed against FGF2, administered immediately before arterial injury, inhibited SMC proliferation measured 41 hours after injury but produced no significant reduction in intimal area at 8 days. This same antibody to FGF2, given 4 and 5 days after injury, resulted in no significant reduction in intimal SMC proliferation.6 These findings suggested that FGF2 influences the proliferative response early but has a lesser role in the continued intimal SMC proliferation that occurs 4 to 14 days after injury.6 In contrast, the present study demonstrates that repeated injections of rFGF2-SAP produce a smaller neointima at 14 and 28 days, suggesting that SMCs continue to respond to FGF2 signals beyond the peak period of SMC proliferation (0 to 4 days) in the long-term neointimal responses to injury. These different results may reflect different experimental approaches (antibody to FGF2 versus FGF2-directed cytotoxicity with rFGF2-SAP) to reduce neointimal growth.
The present study also shows that the SMCs that migrate and proliferate within the injured arterial segment express FGFR1. Intense FGFR1 expression seen on intimal and medial SMCs through at least up to 14 days after injury may explain the long-term suppression of neointimal growth with repeated dosing of rFGF2-SAP. Four classes of FGF receptors have been identified.21 mRNA for FGFR1 has been shown to be present on endothelial cells 8 hours after injury and on both replicating and quiescent SMCs 5 days and 6 weeks, respectively, after injury.7 Casscells et al14 demonstrated binding of radiolabeled FGF2 to medial SMCs 2 days after injury and on neointimal SMCs at 4 days; by immunohistochemistry, there was positive antibody staining for FGFR1 on neointimal SMCs at 14 days. Together with earlier results, finding FGFR1 on medial and intimal SMCs at 3 and 14 days and on adventitial cells on day 3 in the present study suggests that these migrating and proliferating cells are targets for rFGF2-SAP and that reduced neointimal size is due to internalization of the cytotoxin and cell death during the period of intimal growth.
There was a significant (56%) reduction in adventitial cellularity at
3 days in rFGF2-SAP–treated animals. This adventitial effect may also
be important in neointimal growth inhibition because adventitial cells
also possess FGFR1 early after injury (Fig 9C) and may be an additional
source of growth factors and cytokines. The diminished adventitial
FGFR1 staining and cellularity observed in rFGF2-SAP–treated arteries
on day 3 suggests that rFGF2-SAP affects cells besides SMCs involved in
the responses to vascular injury, particularly inflammatory cells, and
that these adventitial cells play an important role in the regulation
of neointimal growth.
The effect of rFGF2-SAP also appeared to be specific for injured arteries, since medial SMC numbers were similar in noninjured arteries in rats that received rFGF2-SAP or vehicle. Our results and those of others7 indicate that uninjured SMCs also express FGFR1 yet are unresponsive to exogenous rFGF2-SAP or exogenous FGF2 until they are injured. The reason for this lack of response despite receptor expression is unclear but may be secondary to receptor upregulation in the injury response. Positive intracellular staining with an antibody to FGF2 has been shown in uninjured arterial SMCs,6 and FGF2 has been shown to be localized in the nucleus.5 22 FGF2 is not secreted via classical signal transduction pathways23 but is believed to be released via a distinct export pathway24 and in the setting of cell injury or death.6 A reservoir of FGF2 may also be present in the extracellular connective tissue matrix,25 26 27 and extracellular FGF2 may increase via the release of intracellular stores as a result of SMC injury.28 Thus, although uninjured quiescent SMCs contain FGF2 and express FGF receptors, they remain unresponsive to stimuli until injured.
The data in the present study suggest that a targeted therapy for reducing SMC number and neointimal formation may be accomplished by using a mitotoxin such as rFGF2-SAP. However, the results also support the hypothesis that therapies that limit restenosis in the short term may only delay neointimal formation; in order to achieve sustained inhibition of neointimal growth, prolonged contact of drug with the previously damaged arterial wall may be necessary. It should be noted that in the present report, the optimal dose of rFGF2-SAP for inhibition of neointima formation was not identified, since higher doses resulted in systemic toxicity. Major targets of toxicity include the liver and kidney, the sites of metabolism and elimination, respectively (authors' unpublished data, 1996). Thus, a shorter duration of treatment with a higher local concentration may result in sustained suppression of neointimal growth. In addition, local delivery of rFGF2-SAP would also be expected to have a favorable influence on the therapeutic index.
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Selected Abbreviations and Acronyms |
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Footnotes |
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Reprint requests to Renu Virmani, MD, Department of Cardiovascular Pathology, Armed Forces Institute of Pathology, Washington, DC 20306-6000.
The opinions and assertions contained herein are the private views of the authors and are not to be construed as official or reflecting the views of the Department of the Army or the Department of Defense.
Received November 7, 1996; accepted December 30, 1996.
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