(Hypertension. 2000;36:364.)
© 2000 American Heart Association, Inc.
Scientific Contributions |
Bradykinin B1 Receptor Mediates Inhibition of Neointima Formation in Rat Artery After Balloon Angioplasty
From the Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston.
Correspondence to Julie Chao, PhD, Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC 29425-2211. E-mail chaoj{at}musc.edu
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Abstract—We evaluated the effects of the kallikrein-kinin system on the proliferation and migration of primary cultured vascular smooth muscle cells (VSMCs) in vitro and neointima formation in balloon-injured rat carotid arteries in vivo. In cultured rat VSMCs, tissue kallikrein inhibited cell proliferation, and this inhibitory effect was blocked by Sar-Tyr-Aca(
)-Lys
[D-ßNal7,Ile8]-des-Arg9-bradykinin,
a bradykinin B1 receptor antagonist, and by
icatibant, a bradykinin B2 receptor antagonist.
Platelet-derived growth factor significantly increased the
expression of the B1 receptor but not the B2
receptor in VSMCs. Platelet-derived growth factor–induced cell
migration was significantly attenuated by
des-Arg9-bradykinin and to a lesser degree by bradykinin.
Endogenous B1 receptor mRNA increased in rat
carotid arteries after balloon angioplasty. After local delivery of
adenovirus carrying the human tissue kallikrein gene into the rat
carotid artery, we observed a 54% reduction in the intima/media ratio
at the injured site compared with the control ratio (n=7,
P<0.01). Administration of the B1 receptor
antagonist via minipumps blocked the protective effect of
kallikrein and partially reversed the intima/media ratio toward the
control ratio. Kallikrein gene delivery results in the regeneration of
endothelium compared with the control groups, and the
B1 receptor antagonist abolished this effect.
Nitrite/nitrate, cGMP, and cAMP levels in balloon-injured arteries
significantly increased after kallikrein gene delivery, whereas the
B1 receptor antagonist abolished these
increases (n=4 or 5, P<0.05). These results indicate
that the B1 receptor contributes to the reduction of
neointima formation via the promotion of
reendothelialization and inhibition of VSMC
proliferation and migration through NO-cGMP and cAMP signaling
pathways. This study provides significant implications in treating
restenosis after revascularization.
Key Words: bradykinin • kallikrein-kinin system • gene delivery • cell migration • neointima formation
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Introduction |
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Balloon catheter injury to the rat carotid artery triggers a sequence of events, including early platelet accumulation, proliferation of medial smooth muscle cells, migration into the intima, and proliferation of intimal cells to form neointima hyperplasia. It was reported that the angiotensin-converting enzyme (ACE) inhibitor has been shown to suppress neointima formation after endothelial injury in the rat carotid artery and abdominal aorta.1 Because angiotensin II exhibits mitogenic activity in vascular smooth muscle cell (VSMC) growth and proliferation,2 it was thought that the inhibition of angiotensin II is related to the suppression of neointima formation by the administration of ACE inhibitors. Inhibition of ACE activity not only prevents the formation of angiotensin II but also increases kinin levels by preventing kinin degradation. Previous studies have shown that icatibant, a bradykinin (BK) B2 receptor antagonist, can partially block the inhibitory effect of ACE inhibitors on neointima formation.3 4 Furthermore, we have recently reported that a continuous supply of tissue kallikrein by gene delivery suppressed neointima formation in the balloon-injured artery and that this effect was inhibited by icatibant.5 These combined results indicate a protective role of kinins in neointima formation after balloon angioplasty.
There are 2 BK receptor subtypes, B2 and B1 receptors. Kinin and its kininase I metabolite, des-Arg9-BK or des-Arg10-Lys-BK, activate B2 and B1 receptors, respectively. The B1 receptor is not expressed or is expressed at low levels in tissues under normal conditions, but it is induced under pathological conditions. It has already been reported that the B1 receptor is induced by cytokines such as interleukin-1ß, bacterial lipopolysaccharides, or vascular injury.6 7 8 9 Induction of B1 receptor–dependent vasoconstriction has also been shown in the rabbit carotid artery after balloon injury.10 In addition, a previous study has shown that the B1 receptor agonist inhibits platelet-derived growth factor (PDGF)-stimulated proliferation in rat mesenteric smooth muscle cells in vitro.11 However, whether B1 receptors affect VSMC migration and/or neointima formation in injured blood vessels after balloon angioplasty has not been investigated. In the present study, we evaluated the potential role of B1 receptors in the migration and proliferation of VSMCs in vitro and neointima formation in the rat artery after balloon angioplasty in vivo by both pharmacological and gene delivery approaches. The present study provides new insights into the role of BK B1 receptors in VSMC growth, migration, and reendothelialization after vascular injury.
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Methods |
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Preparation of Adenovirus Carrying the Human Tissue Kallikrein Gene
Adenovirus containing the human tissue kallikrein gene under the control of the cytomegalovirus promoter (Ad.CMV-cHK) was generated as previously described.12 Large quantities of high-titered adenoviruses, Ad.CMV-cHK and control virus containing the luciferase gene (Ad.CMV-Luc), were prepared and purified for gene delivery.13
Primary Cultured Aortic Smooth Muscle Cell Proliferation
Assay
Rat VSMCs were isolated from the thoracic aorta of male
Sprague-Dawley rats (180 to 200 g, Harlan Sprague Dawley, Inc,
Indianapolis, Ind) by the combined collagenase and
elastase digestion method.14 The cells exhibited a
"hill-and-valley" growth pattern and were characterized by positive
immunostaining with monoclonal antibodies against
smooth muscle 
-actin.15 Cells were serially passaged
and used between passages 3 and 10. Quiescent VSMCs in 24-well plates
were treated with 0.1 µmol/L of rat tissue kallikrein and
Sar-Tyr-Aca()-Lys
[D-ßNal7,Ile8]-des-Arg9-BK,
a BK B1 antagonist kindly provided by
Dr D. Regoli, Institut de Pharmacologie de Sherbrooke,
Canada,16 or icatibant, a BK B2
antagonist kindly provided by Hoechst Roussel
Pharmaceuticals Inc, Germany, at 1 µmol/L in 0.1% FBS DMEM for
18 hours and then pulse-labeled with 1.0 µCi/mL
[3H]thymidine for another 6 hours. At the end
of the incubation, cells were washed 3 times with PBS, precipitated
with 10% trichloroacetic acid for 30 minutes at 4°C, washed 2 times
with 95% ethanol, and then solubilized with 0.25 mol/L NaOH plus 0.1%
SDS. After neutralization with 1 mol/L acetic acid, the radioactivity
was determined by a liquid scintillation counter (Packard). Each
experiment was performed in quadruplicate.
Cell Migration Assay
VSMC cell migration was assessed by using modified Boyden
chambers (Corning Inc).17 18 The transwell inserts
were coated with a solution of 10 µg/mL fibronectin and 50 µg/mL
type I collagen (Sigma Chemical Co) and then air-dried. VSMCs
(2x105 cells) suspended in a 200 µL aliquot of
DMEM containing 0.1% BSA were added to the upper chamber and incubated
in DMEM containing 0.1% BSA for 1 hour. Transwell
apparatuses were then incubated with testing samples for 4
hours at 37°C. Rat PDGF-BB (Sigma) at 15 ng/mL or
des-Arg9-BK or BK (Sigma) at 1 mol/L dissolved in
DMEM containing 0.1% BSA was used as the chemoattractant and was
placed in the bottom chamber of Boyden apparatus. At the
end of incubation period, cells on both sides of the membrane were
fixed and stained with modified Wright-Giemsa stain (Sigma). Cells on
the upper surface of the membrane were then mechanically removed, and
the average number of cells from 5 randomly chosen high-power (x400)
fields on the lower side of the membrane was counted. Each experiment
was performed in triplicate.
Animal Treatment
All procedures complied with the standards for care and use of
animal subjects as stated in the Guide for the Care and Use of
Laboratory Animals (Institute of Laboratory Resources, National
Academy of Sciences, Bethesda, Md). Male Sprague-Dawley rats (weight
350 to 400 g) were anesthetized with ketamine (90
mg/kg IP) and xylazine (10 mg/kg IP), and a 2F embolectomy balloon
catheter (Baxter Health Care Corp) was introduced into the left common
carotid artery by way of the external artery. The balloon was inflated
with sufficient saline to distend the common carotid and was then
withdrawn to the external artery. This procedure was repeated 3 times,
and the catheter was then removed. After balloon injury of the left
common carotid artery, the injured distal segment was isolated by
temporary ligature. The adenoviral particle of Ad.CMV-cHK or Ad.CMV-Luc
(4x109 plaque-forming units in 20 µL) was
infused into the distal injured segment and incubated for 15 minutes at
room temperature. After incubation, the cannula was removed, and blood
flow to the common carotid artery was restored. To investigate the
potential kinin-mediated effect after kallikrein gene delivery,
Sar-Tyr-Aca()-Lys
[D-ßNal7,Ile8]-des-Arg9-BK
(B1 antagonist) was infused
intraperitoneally at a rate of 70 µg/kg per day
after balloon angioplasty and Ad.CMV-cHK infusion. At 7 and 14 days
after gene delivery, rats were anesthetized and perfused with
saline through the ascending aorta. Both carotid arteries were isolated
for nitrite/nitrate (NOx), cGMP, and cAMP assays or morphometric
analysis.
RT-PCR and Southern Blot Analysis
Total RNA was extracted with Trizol reagent according to the
protocol recommended by the manufacturer (BRL). Semiquantitative
reverse transcription–polymerase chain reaction (RT-PCR) and Southern
blot analysis were used to determine the abundance of
B1 receptors and B2
receptor mRNA in nontreated, sham-operated, and injured carotid
arteries at 7 and 14 days after angioplasty in vivo and in quiescent
cultured VSMCs treated with or without PDGF at 15 ng/mL for 30 minutes
in vitro. Specific oligonucleotide probes for the
B1 receptor (5' primer, 5'-AAGACAGCAGTCACCATC-3';
3' primer, 5'-GACAAACACCAGATCGGA-3'; and internal probe,
5'-AAGACTGGGACCTGCTGTAT-3') and the B2 receptor
(5' primer, 5'-GAACATCTTTGTCCTCAGC-3'; 3' primer,
5'-CCGTCTGGACCTCCTTGAAC-3'; and internal probe,
5'-GTAGTCCTTCATGGTCCTGAACACC-3') were used. The quality of RNA was
evaluated by RT-PCR and Southern blot of rat cytoplasmic ß-actin by
using specific primers and internal probe (5' primer,
5'-GAACCCTAAGGCCAACCGTG-3'; 3' primer, 5'-TGGCATAGAGGTCTTTACGG-3'; and
internal probe, 5'-CGCACGATTTCCCTCTCAGC-3').
Assays for NOx, cGMP, and cAMP levels
At 7 days after angioplasty and gene delivery, the rats were
anesthetized, and the carotid artery was dissected and
homogenized (Polytron, Brinkmann Instruments) in 400 µL
of 0.1N HCl at 4°C and centrifuged at 15 000g for 30
minutes. cGMP and cAMP levels were measured by
radioimmunoassay,19 and NOx was measured by a fluorometric
assay as previously published.20 Protein concentrations
were determined by Lowry’s method as previously
described.21
Morphometric Analysis
Two weeks after gene delivery, the rats were
anesthetized, and the left and right carotid arteries were
removed and embedded in paraffin. Each artery was divided into 3
segments, which were separately embedded in paraffin. Cross-sectional
rings (4 µm) were cut from each segment and stained with
hematoxylin and eosin. The slides were photographed with a microscope
at a magnification of x100. The luminal, neointimal, and
medial areas were measured by use of the NIH Image 1.60 software
package. For evaluating recovery of the endothelium,
antibody against von Willebrand factor was used to identify
endothelial cells (Dako). The standard immunoperoxidase
procedure using the avidin-biotin-peroxidase complex (Vectastain ABC
kits, Vector Laboratory) was performed according to the manufacturer’s
instructions. The sections were then developed with 0.02%
H2O2 and 0.1%
diaminobenzidine tetrahydrochloride. The
reendothelialization index was defined as the
percentage of luminal circumference lined by newly regenerated
endothelium in the inner lumen
circumference.22 The mean of the
reendothelialization index was calculated from 3
different cross sections of each artery from 7 rats per group.
Statistical Analysis
Group data are expressed as mean±SEM. Data were compared
between experimental groups by 1-way ANOVA. Differences between
kallikrein and control groups were further evaluated by the Fisher
protected least squares differences. Differences were considered
significant at a value of P<0.05.
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Results |
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Effects of Tissue Kallikrein and B1 and B2 Receptor Antagonists on the Proliferation of Primary Cultured VSMCs
Figure 1 shows that tissue kallikrein (0.1 µmol/L) significantly inhibited the proliferation of VSMCs compared with control proliferation as measured by [3H]thymidine incorporation (67.8±3.6% of control, n=4, P<0.01). The inhibitory effect of tissue kallikrein was abolished by 1 µmol/L of Sar-Tyr-Aca(
)-Lys
[D-ßNal7,Ile8]-des-Arg9-BK,
a B1 receptor antagonist (87.0±7.8%
of control, n=4, P<0.05), or icatibant, a
B2 receptor antagonist (89.0±6.3%
of control, n=4, P<0.05).
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Effects of B1 and B2 Receptor Agonists on
the Migration of Primary Cultured VSMCs
Figure 2 shows that
B1 and B2 agonists have no
effect on VSMC migration in the absence of PDGF. PDGF increased the
migration of VSMCs by 3-fold compared with the control value (105±2
versus 35±4 cells per field, n=3, P<0.01). The
B1 agonist, des-Arg9-BK,
reduced 44% of PDGF-stimulated cell migration (59±1 cells per field,
n=3, P<0.01), whereas the B2 agonist,
BK, had a minor effect, with only a 13% reduction in cell migration
(92±5 cells per field, n=3, P<0.01) (Figure 2).
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Differential Expression of B1 and B2
Receptors After Administration of PDGF In Vitro or Balloon Angioplasty
In Vivo
The expressions of endogenous B1
and B2 receptors were analyzed by RT-PCR
followed by Southern blot analysis with the use of 3
gene-specific oligonucleotides for each transcript.
Figure 3A shows the transcripts of
B1 and B2 receptors in
primary cultured VSMCs with or without PDGF. Expression of the
B1 receptor was significantly increased by PDGF,
whereas no significant change was observed in the expression of the
B2 receptor. Figure 3B shows the
transcripts of B1 and B2
receptors in rat carotid arteries after balloon angioplasty. Expression
of the B1 receptor was significantly increased at
7 days in rats after balloon angioplasty compared with control or
sham-operated rats, whereas the expression of the
B2 receptor was unaltered in the injured carotid
artery. Identical levels of ß-actin were detected among these samples
in both experiments, indicating that the RNA quality of these samples
is internally consistent.
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Effects of Local Kallikrein Gene Delivery and B1
Receptor Antagonists on Neointima Formation in
Rat Artery After Balloon Injury In Vivo
Figure 4 shows typical morphology of
the rat carotid artery 14 days after angioplasty and tissue kallikrein
gene delivery. The artery of a sham-operated rat showed normal
morphology (Figure 4A), whereas angioplasty caused
neointima formation in the artery (Figure 4B).
Kallikrein gene delivery significantly reduced thickening of the
arterial wall (Figure 4C), whereas
B1 antagonist treatment abolished the
protective effect of kallikrein (Figure 4D). Figure 5 shows morphometric analysis of
the intimal area and the intima/media ratio in the carotid artery after
balloon angioplasty. The intimal area in rats receiving
adenovirus-mediated kallikrein gene delivery significantly decreased
compared with that in control rats (cross-sectional area, 62.0±6.0
versus 119.0±6.7 µm2 [mean±SEM],
respectively; n=7; P<0.01). A 54% reduction in the
intima/media ratio was found in rats receiving kallikrein gene delivery
compared with control rats (0.47±0.04 versus 1.03±0.09 [mean±SEM],
respectively; n=7; P<0.01). No significant difference in
intimal area or the intima/media ratio was detected between the
angioplasty groups with or without the control virus containing the
luciferase gene. Suppression of the intimal area and reduction of the
intima/media ratio after kallikrein gene delivery were partially
blocked by the B1 antagonist
(62.0±6.0 µm2 and 0.47±0.04 versus
94.8±6.2 µm2 and 0.82±0.06
[mean±SEM], respectively; n=7; P<0.01). There were still
significant differences in intimal area and the intima/media ratio
between the group receiving Ad.CMV-cHK with the
B1 antagonist and the control group
(P<0.05). No significant difference in the medial area was
found among these groups.
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Effects of Local Kallikrein Gene Delivery and B1
Receptor Antagonists on
Reendothelialization in Rat Artery After Balloon Injury
In Vivo
Figure 6 shows
reendothelialization 14 days after balloon angioplasty
among the different groups. The index of
endothelialization was 37.7±13.7% and 35.6±6.9%,
respectively, in balloon-injured arteries of rats with and without
Ad.CMV-Luc (n=7 in each group). A prominent recovery of
endothelium was observed in the balloon-injured
arteries of rats receiving kallikrein gene delivery compared with
control group arteries (88.6±5.8% versus 37.7±13.7% and
35.6±6.9%, n=7, P<0.01). Administration of the
B1 antagonist abolished the
kallikrein-mediated promotion of reendothelialization
(57.7±10.9% versus 88.6±5.8%, n=7, P<0.05).
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Effects of Kallikrein Gene Delivery and B1 Receptor
Antagonists on NOx, cGMP, and cAMP Levels in the Carotid
Artery After Balloon Angioplasty
Figure 7 shows the effect of
kallikrein gene delivery and B1 receptor
antagonist on NOx, cGMP, and cAMP levels in the carotid
artery after balloon angioplasty. NOx content in the artery increased
significantly in the group receiving the adenovirus containing the
tissue kallikrein gene versus the angioplasty group and the group that
received Ad.CMV-Luc (225.4±32.8 versus 94.8±27.0 and 68.7±37.4
pmol/mg protein, n=4 or 5, P<0.05). Administration of the
B1 antagonist inhibited the increase
of NOx content (80.4±28.2 pmol/mg protein) (Figure 7A). cGMP
levels in the carotid artery increased significantly in the group
receiving the adenovirus containing the kallikrein gene compared with
the angioplasty groups with and without Ad.CMV-Luc (32.9±2.3 versus
18.9±2.0 and 20.9±4.9 pmol/mg protein, n=4 or 5, P<0.05).
Administration of the B1 antagonist
inhibited the increase of cGMP (19.5±4.6 pmol/mg protein) (Figure 7B). Similarly, cAMP levels in the artery increased
significantly in the group receiving the adenovirus containing the
kallikrein gene compared with the angioplasty groups with and without
Ad.CMV-Luc (581.9±67.2 versus 376.6±51.6 and 339.4±79.4 pmol/mg
protein, n=4 or 5, P<0.05). Administration of the
B1 antagonist inhibited the increase
of cAMP (303.4±43.4 pmol/mg protein) (Figure 7C).
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Discussion |
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This is the first study to demonstrate that the BK B1 receptor plays an important role in the reduction of neointima formation after balloon angioplasty. Local delivery of the tissue kallikrein gene at the injured site results in significant reduction of the intima/media ratio and neointima formation as well as prominent recoveries of endothelium and increases of NOx, cGMP, and cAMP levels at the injured site. The B1 receptor antagonist abolished the protective effects of kallikrein on arterial thickening and the increases in vascular NOx, cGMP, and cAMP levels. In primary cultured VSMCs, the B1 antagonist abolished the inhibitory effect of kallikrein on cell proliferation, and the B1 receptor agonist inhibited PDGF-stimulated VSMC migration. These results indicate that the BK B1 receptor attenuates neointima formation in the balloon-injured artery through promotion of reendothelialization and inhibition of VSMC migration and proliferation via activation of NO-cGMP and cAMP signaling pathways. These findings provide important insights into the role of the BK B1 receptor in occlusive vascular diseases.
Oza et al23 reported that kininogen is synthesized in cultured rat aortic smooth muscle cells. Our previous study has also shown the expression of endogenous kininogen and tissue kallikrein in rat artery and aorta by RT-PCR Southern blot analysis by using specific oligonucleotide probes.5 Furthermore, expression of the B1 receptor has been detected in VSMCs.24 In the present study, we showed that B1 receptor mRNA was increased in primary cultured VSMCs after PDGF treatment in vitro (Figure 3A). Consistent with the in vitro data, we found that the expression of the endogenous B1 receptor was increased in the rat carotid artery 1 week after balloon injury (Figure 3B). Together, these results demonstrate that all of the key components of the kallikrein-kinin system are present in VSMCs, where they may function by autocrine/paracrine mechanisms.
Our present study has shown that adenovirus-mediated delivery of the tissue kallikrein gene into the rat artery attenuates neointima formation after balloon injury and that the B1 receptor antagonist partially reverses this effect (Figure 5). This result suggests that the BK B1 receptor, induced by balloon angioplasty, contributes to the inhibition of neointima formation. To investigate the mechanisms of the BK B1 receptor on the inhibition of neointima formation, we performed cell culture studies. VSMC proliferation is one of the important components of neointima formation after balloon angioplasty. Dixon and Dennis11 showed that the B1 or B2 agonist inhibited PDGF-induced cultured arterial smooth muscle cell proliferation. We found that tissue kallikrein suppressed VSMC proliferation in vitro and that this effect was blocked by the B1 or B2 antagonist (Figure 1). These data suggest that the inhibitory effect of kallikrein on VSMC proliferation is mediated by local release of kinin or kinin metabolites in VSMCs, as carboxypeptidase M, which converts BK to des-Arg9-BK, has been demonstrated in VSMCs.25 On the other hand, VSMC migration is another important component of neointima formation after balloon angioplasty.26 It has been well established that the expression of PDGF increased markedly in injured tissues and that this growth factor plays an important role in VSMC migration and growth after balloon injury.27 28 29 In the present study, we showed that the B1 receptor plays a major role in the inhibition of PDGF-stimulated cell migration but that the B2 receptor has only a minor effect. The B1 agonist reduced 44% of PDGF-stimulated cell migration, whereas the B2 agonist reduced only 13%, and the BK B1 or B2 agonist alone did not have any effect on VSMC migration in the absence of PDGF (Figure 2). These results suggest a synergistic action of the B1 agonist and PDGF. Another potential mechanism for the inhibition of neointima formation is via reendothelialization, which leads to recovery of beneficial functions of the endothelium.22 The key role of the endothelium in regulating underlying intimal growth is well known, and endothelium has a major influence on the degree of intimal hyperplasia.30 31 We showed that kallikrein gene delivery accelerated the growth of endothelium in the injured artery and that the B1 antagonist abolished this effect (Figure 6). This finding is consistent with previous reports that the B1 receptor can induce endothelial cell proliferation and angiogenesis.32 33 Taken together, our results indicate that activation of the B1 receptor has multiple effects on neointima formation, including inhibitory effects on both migration and proliferation of VSMCs as well as the promotion of reendothelialization, whereas the B2 receptor mainly inhibits VSMC proliferation. However, we found that the inhibitory effect of the B2 receptor antagonist against kallikrein gene delivery appears to be stronger than that of the B1 receptor antagonist,5 because it must be noted that the B1 receptor is induced after vascular injury, whereas the B2 receptor is constitutively expressed. Moreover, the effect of kallikrein gene delivery is completely blocked by the NO synthase (NOS) inhibitor, inasmuch as both effects of B1 and B2 receptors are mainly mediated by NO.34
It has been reported that the overexpression of human
endothelial NOS mediates the decrease of
neointima formation after balloon injury35 and
that NO inhibits smooth muscle cell growth through the cGMP signaling
pathway.36 Our results have shown that local kallikrein
gene delivery into the carotid artery after angioplasty results in
increased NOx and cGMP levels at the injured vessel and that the
B1 antagonist suppresses these
elevations. These findings suggest that the inhibitory
effect of neointima formation after kallikrein gene
delivery is in part mediated by B1
receptor–NO–cGMP signaling pathways. Previous reports have shown that
the NOS inhibitor
N
-nitro-L-arginine
methyl ester could block the beneficial effect of ACE
inhibition4 and kallikrein gene
delivery34 on neointima formation in
balloon-injured rat carotid arteries. Also, BK significantly increased
nitrite release from isolated canine coronary microvessels, and
the increased release of nitrite was dramatically reduced with NOS
inhibition.37 In the present study, we showed that the
B1 antagonist abolished the elevation
of NOx and cGMP levels in the carotid artery after kallikrein gene
delivery. These findings indicate that the metabolites of kinin may
stimulate NO and cGMP production through the activation of NOS
via the BK B1 receptor. One potential source of
NO may be the regenerated endothelium, because 
90%
of the neointima is covered with newly formed
endothelial cells 14 days after kallikrein gene
delivery and balloon angioplasty (Figure 6), and NO can be
produced via endothelial NOS. Another source of NO may
be inducible NOS from VSMCs. This notion is supported by a report that
arterial smooth muscle cells express inducible NOS in
response to endothelial injury.38 Elevated
cGMP formation regulates the cell proliferation and migration through
cGMP-mediated activation of tyrosine phosphatases,39
cGMP-dependent protein kinase,40 41 42 43 or cross-activation
of cAMP-dependent protein kinase.44
Prostacyclin activates adenylate cyclase, leading to increased cAMP levels. Elevation of cAMP production attenuates mitogen-activated protein kinase signaling induced by PDGF in VSMCs and thus inhibits cell proliferation.45 Furthermore, elevated cAMP production also suppresses VSMC migration via protein kinase A.41 42 43 Interestingly, we found that vascular injury increased cAMP production in the carotid artery (Figure 7C). We speculate that inflammation may cause cAMP elevation by means of stimulation of prostaglandin production,46 and elevated levels of cAMP could contribute, in part, to self-protection for reduced neointima formation. Our results also showed that kallikrein gene delivery significantly increased cAMP levels in the carotid artery and that the B1 antagonist blocked this elevation. These results suggest that the inhibitory effect of neointima formation after kallikrein gene delivery is, in part, mediated by B1 receptor–cAMP signaling pathways, because binding of kinin metabolites to the B1 receptor stimulates the production of prostacyclin.47 Detailed mechanisms by which the B1 receptor contributes to the inhibition of VSMC proliferation and migration remain to be further elucidated.
In conclusion, using pharmacological and gene delivery approaches, we showed that the BK B1 receptor contributes to the attenuation of neointima formation after balloon injury through the promotion of reendothelialization and the inhibition of VSMC proliferation and migration via NO-cGMP and cAMP signaling pathways. These results provide new insight into the role of the vascular BK B1 receptor and have significant implications for gene therapy in the treatment of restenosis and arteriosclerosis.
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Acknowledgments |
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This work was supported by National Institutes of Health grants HL-52196 and HL-29397.
Received December 21, 1999; first decision January 21, 2000; accepted April 11, 2000.
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References |
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