© 1996 American Heart Association, Inc.
Articles |
Expression of Endothelin-1, Endothelin-3, Endothelin-Converting Enzyme-1, and Endothelin-A and Endothelin-B Receptor mRNA After Angioplasty-Induced Neointimal Formation in the Rat
From the Departments of Cardiovascular Pharmacology (X.W., S.A.D., L.M.V.-C., G.Z.F., E.H.O.) and Experimental Pathology (C.L.), SmithKline Beecham Pharmaceuticals, King of Prussia, Pa.
Correspondence to Dr Xinkang Wang, Department of Cardiovascular Pharmacology, SmithKline Beecham Pharmaceuticals, PO Box 1539, UW2511, 709 Swedeland Rd, King of Prussia, PA 19406.
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Abstract |
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Abstract Endothelins (ETs) are potent vasoconstrictors known to play a role in tissue remodeling after vascular wall injury. The molecular mechanisms for the expression and functions of ETs and their receptors after carotid artery angioplasty are not fully understood. Using quantitative reverse transcription and polymerase chain reaction, the present study demonstrates the temporal mRNA expression of ET-converting enzyme-1 (ECE-1), preproET-1, preproET-3, and both ETA and ETB receptors after rat carotid artery balloon angioplasty. A significant increase in ECE-1 mRNA was observed at 6 hours (1.8-fold increase over control, P<.01) and 24 hours (1.7-fold increase, P<.01) in carotid arteries after angioplasty. In contrast, a significant increase in preproET-1 mRNA levels was not observed until 3 days (1.9-fold increase, P<.05) and 7 days (2.1-fold increase, P<.05). A similarly delayed increase in preproET-3 mRNA was observed at 7 days (2.8-fold increase, P<.05) and 14 days (2.6-fold increase, P<.05) after angioplasty. A parallel but marked increase in ETA and ETB receptor mRNAs compared with preproET-1 and -3 messages was observed after angioplasty. The levels of ETA receptor mRNA were elevated 29.3-fold (P<.001) and 24.3-fold (P<.01) at 3 and 7 days, respectively, after angioplasty. The increase in ETB receptor mRNA occurred slightly earlier than the increase in ETA receptor mRNA, showing 15.1-fold increase at 1 day (P<.001) and 11.3-fold increase at 3 days (P<.01) after angioplasty. Immunohistochemical studies using anti-ET antibodies demonstrated a corresponding increase in ET immunoactivity, which was distributed mainly in the neointimal cells 14 days after angioplasty. The increases in ECE-1, ET-1, and ET-3 and their receptor expression after balloon angioplasty suggest that these proteins play an active role in the pathogenesis of neointimal formation.
Key Words: endothelin system • neointimal formation • balloon angioplasty • reverse transcription/polymerase chain reaction
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Introduction |
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Endothelins (ETs) are a family of 21-amino-acid peptides, including ET-1, ET-2, and ET-3, in mammals. ET-1 is the most potent mammalian vasoconstrictor identified to date.1 In addition to controlling vascular smooth muscle tone, ET-1 also influences the tissue remodeling process following vascular wall injury. In vitro, ET-1 stimulates cellular hypertrophy and hyperplasia, matrix synthesis, chemokinesis, and the release/expression of several chemotactic/growth factors and adhesion molecules.2 3 One procedure associated with pronounced vascular wall trauma is PTCA, a surgical procedure developed to alleviate the myocardial ischemia associated with the presence of advanced occlusive atherosclerotic human coronary artery lesions.4 However, the long-term efficacy of PTCA is severely limited by the high incidence of vascular restenosis, the result of occlusive fibroproliferative intimal lesion formation within the vessel lumen. Therefore, it is interesting to note that clinical evidence exists to demonstrate activation of the ET system both in the development of primary atherosclerotic lesions5 and after PTCA,6 7 situations in which ET-1–like immunoreactivity is elevated in the human coronary sinus (possibly as a result of both endothelial cell stretch/damage and de novo peptide synthesis). cDNA sequence analysis suggests that the primary translational product of the human ET-1 gene is a 212-amino-acid prohormone, preproET-1. It has been proposed that preproET-1 is catabolized by a dibasic-pair–specific endopeptidase to produce a 38-amino-acid peptide, an inactive molecule termed big ET-1. This peptide intermediate undergoes further proteolytic maturation (Trp21-Val22 cleavage) by a novel neutral endopeptidase, ECE-1, to produce the biologically active ET-1.8 9 Two subtypes of ET receptors have been identified and characterized: ETA and ETB receptors.10 11 Both of these receptors are expressed on vascular smooth muscle cells and mediate the direct vasoconstrictor action of ETs. In addition, both receptors are also actively involved in mediating a variety of biological actions based on their G protein–coupled signal transduction pathways.
One preclinical model that has been developed to study the role of putative pathogenic mediators involved in the phenomenon of restenosis is the rat carotid artery balloon angioplasty model. Administration of exogenous ET-1 augments neointimal formation in this model.12 13 Furthermore, immunohistochemical studies show that endogenous ET-1 levels are elevated within the wall of rabbit carotid arteries 1 to 4 weeks after angioplasty.14 Indeed, since chronic administration of an ET receptor antagonist SB 209670 is vasculoprotective in the rat, it has been proposed that such elevations appear to be of pathological significance.12 This antagonist exhibits high affinity for both the ETA and the ETB receptor.15 In contrast, the ETA-selective antagonist BQ-123 is devoid of vasculoprotective efficacy in either the rat16 or the rabbit14 carotid artery angioplasty models.
In view of the uncertainties regarding the extent to which the ET system is activated after angioplasty, the aim of the present study was to examine the relative temporal expression of mRNAs encoding for ECE-1, ET-1, ET-3, and the two ET receptors by means of a quantitative RT/PCR method using rpL32 as an internal control for coamplification.17 This method was chosen because of the relatively low abundance of the messages and limited amount of tissue available for the balloon catheter–induced injury of the rat carotid artery.
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Materials and Methods |
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TopAbstractIntroductionMaterials and Methods Results Discussion References |
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Left Common Carotid Artery Balloon Angioplasty
Left common carotid artery balloon angioplasty was performed on male Sprague-Dawley rats (400 g, Charles River, Wilmington, Mass) under sodium pentobarbital anesthesia (65 mg/kg IP) as described previously.2 Briefly, after an anterior midline incision, the left external carotid artery was identified and cleared of adherent tissue, allowing the insertion of a 2F Fogarty arterial embolectomy catheter (Baxter Healthcare Corp). The catheter was guided a fixed distance (5 cm) down the common carotid artery to a point such that the tip of the catheter was proximal to the aortic arch, where the balloon was inflated and withdrawn back to its point of insertion. This procedure was performed a total of three times, after which the catheter was removed and a suture was tied around the external carotid artery to prevent exsanguination. Finally, the wound was closed using 9-mm Autoclips (Clay Adams), and the surgical area was swabbed with Povadyne surgical scrub (7.5% Povidone-Iodine, Chaston). Throughout the surgical procedure, body temperature was maintained at 37±1°C using a K-20-F water blanket (American Hamilton).
Animals were allowed to recover from surgery and were housed in pairs in Plexiglas cages on 12-hour light/dark cycles with access to standard laboratory chow and drinking water ad libitum. All surgical interventions were performed in accordance with the guidelines of the animal care and use committee, SmithKline Beecham, and the American Association for Laboratory Animal Care.
Isolation of Common Carotid Arteries
Left common carotid
arteries were isolated from rats immediately
after exsanguination under sodium pentobarbital anesthesia
(65 mg/kg IP, 65 mg/kg IV bolus). Vessels were removed at the following
time points: 0 hours (control), 6 hours, and 1, 3, 7, and 14 days after
surgery. Once isolated, vessels were immediately frozen in liquid
N2 and stored at -70°C for RNA preparation. Each
time point consists of vessels pooled from three rats (per RT/PCR
analysis), and five separate pooled samples were
analyzed to obtain the statistical data.
RT/PCR
Total cellular RNA was isolated using an acid
guanidinium–phenol–chloroform extraction
procedure.18 Quantitative RT/PCR was carried out
essentially as described in detail previously.17 Briefly,
total RNA (3 µg per sample) was used for RT in the presence of 200 U
of RNase H- SuperScript II reverse transcriptase
(GIBCO BRL) and 1 µg of
oligo(dT)12-18 primer at 37°C for
60 minutes according to the manufacturer's specification. The
resultant cDNA products were isolated by phenol-chloroform
extraction followed by ethanol precipitation. The cDNA pellets were
resuspended in 120 µL TE (10 mmol/L Tris-HCl and 1 mmol/L EDTA, pH
7.5) and stored at -20°C until required for PCR.
PCR primers
(Table
) were designed on the basis of
published rat cDNA sequences for ECE-1,8
preproET-1,19 preproET-3,20 ETA
receptor,11 ETB receptor,21 and
rpL32.22 The rat rpL32 mRNA was used as an internal
control for the coamplification, since pilot studies demonstrated that
carotid artery balloon angioplasty had no effect on the expression of
rpL32 mRNA. In order to define the optimal amplification conditions, a
series of pilot studies were performed using various amounts of RT
products from 25 to 800 ng RNA and 15 to 40 cycles of PCR
amplification in the presence of various amounts of
32P-labeled primers as described previously.17
A set of the representative data showing the
coamplification of preproET-1 and rpL32 mRNA is illustrated in Fig
1. On the basis of these initial experiments, the linear
portion of the amplification was determined for both the testing genes
and the internal standards (ie, rpL32 in the present study).
Therefore, the following conditions were chosen as standard for the PCR
reactions in a volume of 50 µL: RT products from 100 ng RNA, 2.5
U TaqAmpli polymerase (Perkin-Elmer Centus), 30 cycles of amplification
for ECE-1, preproET-1, and ETB receptor genes, or 40 cycles
for preproET-3 and ETA receptor genes, in the presence of
1x106 cpm (
10 ng) labeled antisense primers for
the examined genes and 5x104 (for 30 cycles) or
1x104 cpm (for 40 cycles) for rpL32 antisense primer
together with 100 ng of each nonradioactive sense and antisense primers
(Table). The amplification was carried out as follows: the
initial
cycle using 3 minutes at 94°C for denaturation, 1 minute at 54°C
for annealing, and 3 minutes at 72°C for extension. Subsequent cycles
of PCR were performed using the following conditions: denaturation, 15
seconds at 94°C; annealing, 20 seconds at 54°C; and extension, 1
minute at 72°C.
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Quantification of PCR Products
PCR products (10 µL per
lane) were electrophoresed using
either 8 mol/L urea with 6% polyacrylamide denaturing gel or
with 6% native polyacrylamide gel. The gel was dried and
subjected to autoradiography at room temperature
overnight. The identity of amplified cDNA products was confirmed by
DNA sequence analysis.
The band intensities were measured using a PhosphorImager with an ImageQuant software package (Molecular Dynamics). The signals of the examined cDNAs were expressed relative to the intensity of rpL32 cDNA in each coamplified sample.
Immunohistochemistry
The carotid artery at 14 days after
balloon angioplasty or
vehicle from five rats was perfusion-fixed with 10%
phosphate-buffered formalin, excised, and stored in formalin. After
24 hours, the artery was transferred to 70% ethanol and subjected to
standard histological processing using a vacuum
infiltration processor (Miles). Before embedding, the middle third
portion of the artery was divided into four equal cross-sectional
segments. Five-micron sections were cut and stained with
hematoxylin and eosin and evaluated microscopically. Additional 5-µm
sections were placed on Capillary Gap Plus microscope slides (BioTek)
for immunohistochemical evaluation of ET expression. The slides were
deparaffinized, rehydrated, placed in a microwave buffer (BioTek), and
microwaved twice for 5 minutes. Sections were stained using an ABC
method on the TechMate automatic stainer (BioTek). Slides were
incubated in primary antibody, a monoclonal antibody at a dilution of
1:100, or a polyclonal antibody against ET-1, which can cross-react
with ET-3 (Biodesign International) at 1:750 dilution, overnight at
4°C to reduce background staining. An absorbed biotinylated secondary
rat antibody at 1:200 dilution was used to reduce cross-reactivity.
As the negative control, the first antibody was incubated with 10 µg
ET-1 peptide (American Peptide) for 1 hour before its addition.
Sections were stained using the ABC method on the TechMate automatic
stainer). After completion of the ABC technique,
3'-3'-diaminobenzidine
was used as the chromagen. Slides were counterstained with hematoxylin,
dehydrated through alcohol, and cleared in xylene before coverslipping.
The slides were evaluated microscopically.
Statistical Analysis
Data are expressed as mean±SE.
Statistical comparisons were
made by ANOVA (Fisher's protected least squares difference), and
values were considered to be significant at P<.05.
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Results |
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Representative autoradiographs of the quantitative RT/PCR experiment to detect the temporal expression of ECE-1, preproET-1, and preproET-3 mRNA in the carotid arteries after balloon angioplasty are shown in Fig 2
, and the corresponding
quantitative data (n=5), after normalizing to the internal control,
rpL32, are illustrated in Fig 3. As shown in Figs
2 and 3, the basal mRNA levels (control samples)
for ECE-1, preproET-1, and
preproET-3 were very low, although the intensity of the relative mRNA
levels for each gene was different. The levels of the mRNA expression
for ECE-1, preproET-1, and preproET-3 were significantly increased as a
result of balloon angioplasty. There were 1.8-fold (P<.01)
and 1.7-fold (P<.01) increases in ECE-1 mRNA levels at 6
and 24 hours after angioplasty, respectively. A significant increase in
the preproET-1 mRNA level after balloon injury was not observed until 3
days (1.9-fold increase over control, P<.05) and 7 days
(2.1-fold increase, P<.05). Similarly, a delayed increase
in preproET-3 mRNA was observed at 7 days (2.8-fold increase,
P<.05) and 14 days (2.6-fold increase, P<.05)
after angioplasty. It was noted that the levels of increases in
preproET-3 mRNA after angioplasty were very low, since 40 cycles were
required for the amplification of this transcript.
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Fig 4 illustrates the representative
autoradiographs of ETA and ETB receptor mRNA
expression in carotid arteries after balloon angioplasty using the
quantitative RT/PCR technique. Very low basal levels of mRNA expression
for both genes were observed in carotid (control) arteries (Figs
4 and 5). The level of ETA receptor
transcript was
increased at 1 day (6.7-fold increase of the mean value compared with
control), then reached a peak level at 3 days (29.3-fold increase,
P<.001) and 7 days (24.3-fold increase, P<.01),
and maintained an elevated level up to 14 days (7.2-fold increase)
after balloon angioplasty (Figs 4 and 5). The
upregulated expression of
ETB receptor mRNA occurred earlier than that of
ETA receptor mRNA, since a marked increase for
ETB receptor transcript was observed 6 hours after
angioplasty. The level of ETB receptor mRNA was elevated
5.5-fold (over control) at 6 hours after angioplasty, reached a maximal
level at 1 day (15.1-fold increase, P<.001) and at 3 days
(11.3-fold increase, P<.01), and then decreased to slightly
elevated levels by 7 days (5.1-fold increase) and 14 days (4.8-fold
increase) in the carotid arteries after balloon angioplasty (Figs
4 and 5). Comparatively, the increases in
ETA/B receptor mRNA (up
to 30-fold increases) were much greater than those of ECE-1 and
preproET-1/3 (2- to 3-fold increases) (Figs 3
and
5).
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In the carotid artery at 14 days after balloon angioplasty, the
hyperplastic neointima exhibited intense positive staining
in a large number of cells when polyclonal anti-ET antibodies were used
(Fig 6). The positive-staining cells were
spindle-shaped with phenotypic characteristics of smooth muscle
cells. With the polyclonal ET antibody that recognizes ET-1 and ET-3,
staining in the hyperplastic neointima revealed a diffused
pattern, with an intensive signal in those cells closest to the lumen
of the ballooned carotid artery. Cytoplasmic and some nuclear staining
was observed in neointimal cells. In the media, only a few
smooth muscle cells exhibited intense cytoplasmic staining after 14
days of balloon angioplasty, and the number of positive-staining
cells in the media was markedly less when compared with the
neointima (Fig 6C). Similar immunostaining
patterns were observed using a monoclonal anti–ET-1 antibody (data not
shown). To confirm the specificity of our observations, the carotid
artery sections 14 days after balloon angioplasty were incubated with
the polyclonal anti-ET antibodies that had been absorbed with ET-1 (Fig
6B). In addition, the time-matched (14 days) but nonballooned
samples were incubated with the primary and secondary antibodies (Fig
6A). As predicted, no positive immunostaining of ET was
detected in both the negative controls.
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Discussion |
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TopAbstractIntroductionMaterials and Methods Results Discussion References |
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In the present report, we demonstrated the temporal expression profiles of ECE-1, preproET-1, preproET-3, and ETA and ETB receptor mRNA after rat carotid artery balloon angioplasty, which were in accord with their biological actions. For example, a significant increase in ECE-1 mRNA occurred at 6 and 24 hours after angioplasty (Figs 2
and 3), which
preceded that of
preproET-1 and preproET-3 mRNAs (at 3 to 7 days for preproET-1 and 7 to
14 days for preproET-3). The significantly upregulated expression of
ETA and ETB receptor mRNAs was in parallel with
that of the ETs (Figs 4 and 5), suggesting that
the enhanced ET action
occurs in the neointimal formation. The demonstration of increased levels of preproET-1 mRNA and peptide after balloon angioplasty is in agreement with the clinical findings in which PTCA6 7 and atherosclerotic plaque23 have been shown to be associated with elevated ET-1–like immunoreactivity. Moreover, the present work represents the first demonstration to show that the increases in ECE-1 mRNA levels are associated with a pathological process. The present findings are of interest in light of the recent observations that both acute and chronic administration of exogenous ET-1 augments lesion formation after balloon angioplasty in the rat,12 24 and they indicate that the changes reported in the present study may be of pathological significance. Furthermore, not only are these findings consistent with immunohistochemical studies performed in rabbit carotid artery models,14 they also corroborate the previous observation that chronic administration of the dual ETA/B receptor antagonist SB 209670 ameliorates neointimal formation in this model.12
It also should be pointed out that under normal conditions, ECE-1 and
ET-1 are synthesized primarily in the
endothelium8 9 25 and not in the media of
blood vessels. However, balloon angioplasty results in immediate and
complete destruction of the endothelium. Therefore, it
is likely that the enhanced expression of ECE-1 and preproET-1 mRNA is
produced by other cellular sources in the neointimal
lesion. In fact, immunostaining demonstrated in the
neointimal cells a high level of ET immunoreactivity with
phenotypic characteristics of smooth muscle cells (Fig 6).
However,
factors that mediate the increased expression of ECE-1 and ET-1 after
angioplasty are unknown, although previous studies demonstrated that
preproET-1 mRNA transcription and secretion of ET-1 in cultured smooth
muscle cells in response to transforming growth factor-ß,
platelet-derived growth factor, platelets,
angiotensin II, and ET-1 itself have been implicated in the
pathogenesis of restenosis.3 26 27
The roles of the ETA and ETB receptors in
mediating the modulatory actions of ET-1 on vascular growth/structure
have been investigated. For example, ET-1–induced cultured vascular
smooth muscle cell proliferation is mediated by either the
ETA or the ETB receptor
subtype,12 28 29 depending on smooth
muscle
phenotype (ie, contractile versus synthetic). Since message
expression of both receptor subtypes is upregulated after angioplasty,
both the ETA and the ETB receptor subtypes may
be equally important for their functions after angioplasty. Moreover,
because different temporal induction profiles and quantitative
differences were noted (Fig 5), the timing of specific
therapeutic
intervention may be important.
Although the upregulation of the transcripts encoding ET system after
balloon angioplasty is evident, it also should be pointed out that the
RT/PCR detection method used in the present report only reflects
the relative quantification, since the amplification rates for the ET
system and the reference gene (ie, rpL32) are not identical (Fig
1). In
numerous cases, similar observations have been reported using a
housekeeping gene as an internal control (such as the use of aldolase
A, ß-actin, histone H3.3, and rpL32) for the
coamplification.17 30 31 32
The different amplification rates
are likely to be caused by (1) different abundances of the messages to
be coamplified, (2) different templates to be used, and (3) different
primers applied. To exclude the possibility that these different rates
may be caused by the different primers used in the present study,
the primers were synthesized with a similar annealing temperature and
were tested for their noninterference between the coamplified primer
sets. On the other hand, different templates may somehow contribute to
different amplification rates, but this should not be the main reason,
since similar approaches (ie, the use of different templates) have been
successfully used in competitive RT/PCR.33 The abundance
of the coamplified messages may, in some degree, contribute to the
different rates of amplification. For these reasons, comparing all the
quantitative PCR techniques used to date, the competitive PCR
technique,34 35 36 37 which
uses only one pair of primers to
amplify the mRNA of interest and an internally deleted version of the
same gene, appears to be advantageous. In that method, the
reference template is synthesized in vitro and quantitatively
distributed into each sample before the amplification. One disadvantage
of this method, however, is its inability to correct the differences
between each sample started for RT. Although some disadvantages may
exist in the PCR quantification using a housekeeping gene as a
reference, relative mRNA quantification should still be able to be
determined if linear amplification is monitored and used for both the
genes of interest and the housekeeping
genes.17 30 31 32
In summary, it appears that ET-1 is implicated in the pathogenesis of angioplasty-induced neointimal lesion formation in the rat carotid artery since (1) acute administration of exogenous ET-1 augments neointimal formation, (2) chronic administration of an ETA/B antagonist ameliorates lesion formation, (3) preproET-1 mRNA and ET-1 immunoreactivity are elevated as a consequence of balloon injury, and (4) ECE-1 mRNA levels are enhanced as a result of vascular wall trauma. Although the precise mechanism by which ET-1 facilitates lesion formation and the receptor subtypes involved in mediating such processes remain to be fully elucidated, the present data suggest that the development of the antagonists for both receptor subtypes may be useful as therapeutic agents.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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The authors wish to express their sincere gratitude to Y. Mao and G. Sathe of the Department of Molecular Genetics, SmithKline Beecham, for oligonucleotide primer synthesis.
Received May 18, 1995; accepted November 1, 1995.
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References |
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