(Hypertension. 2000;35:648.)
© 2000 American Heart Association, Inc.
Scientific Contributions |
Elevated Perfusion Pressure Upregulates Endothelin-1 and Endothelin B Receptor Expression in the Rabbit Carotid Artery
From the Department of Cardiovascular Physiology, University of Goettingen, Germany.
Correspondence to Markus Hecker, PhD, Department of Cardiovascular Physiology, University of Goettingen, Humboldtallee 23, 37073 Goettingen, Germany. E-mail hecker{at}veg-physiol.med.uni-goettingen.de
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
Abstract—To investigate the hypothesis that high blood pressure activates the endothelin system in the vessel wall, isolated segments of the rabbit carotid artery were subjected to different levels of perfusion pressure. Both preproendothelin-1 (ppET-1) mRNA abundance and intravascular ET-1 peptide content were strongly upregulated on raising the intraluminal pressure from 90 to 160 mm Hg for 3 to 12 hours, and this increase in ppET-1 mRNA occurred predominantly in the endothelial cells. Endothelin-converting enzyme-1 and endothelin A receptor (ETA-R) expression were pressure-insensitive, whereas that of the ETB-R in the smooth muscle cells was also significantly enhanced. Both the pressure-induced increase in ppET-1 and ETB-R expression required RNA synthesis because they were abolished by actinomycin D. The nuclear signaling mechanisms involved therein, however, appeared to be different. Thus, the pressure-induced expression of ppET-1 and activation of CCAAT-enhancer binding proteins ß and
were blocked
by the tyrosine kinase inhibitor herbimycin A, whereas
ETB-R expression and the nuclear translocation of
activator protein-1 were abolished by the protein kinase C
inhibitor Ro 31-8220. One consequence of these presumably
deformation-induced changes in gene expression was an increased rate of
apoptosis of the smooth muscle cells in the media that if
transferable to the situation in human blood vessels may contribute to
hypertension-induced arterial remodeling.
Key Words: hypertension, arterial • endothelin • receptors • genes • carotid arteries
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Pressure overload of the vessel wall has been implicated in hypertension-induced arterial remodeling, ultimately leading to manifest hypertension.1 In resistance vessels, remodeling is mainly associated with medial hyperplasia, whereas in conduit arteries it is characterized by medial hypertrophy and enhanced matrix protein synthesis. Among the various mediators implicated in these adaptive processes, endothelin-1 (ET-1) may play a pivotal role.2
Predominantly formed by endothelial cells (EC), this 21–amino acid peptide is not only a powerful vasoconstrictor but also a potent mitogen for vascular smooth muscle cells (SMC). It is derived from a 212–amino acid precursor, prepro-ET-1 (ppET-1), which is sequentially processed to big ET-1 and ET-1 by a furinlike protease and an endothelin-converting enzyme (ECE-1).2 3 ET-1 exerts its biological effects mainly through activation of 2 types of G-protein–coupled receptors, ETA-R and ETB-R.4 Although arterial SMC express both types of receptors, ETA-R activation primarily modulates their tone and growth, whereas the functional significance of the ETB-R in these cells remains to be properly defined.2 3 4
Cyclic strain has been reported to enhance ET-1 peptide synthesis and ppET-1 mRNA expression in cultured EC.5 6 In the vessel wall in situ, EC are not normally exposed to this hemodynamic force because the bulk of the physiological increase in transmural pressure is transformed into a circumferential tensile strain that almost exclusively affects the SMC.7 However, in situations in which the pressure-induced distention of the vessel wall is more pronounced and/or chronically elevated, as in arterial hypertension, EC may also be deformed to a significant extent. To delineate as to whether a supraphysiological increase in (blood) pressure affects ppET-1 gene expression also in the vessel wall in situ, we have developed an experimental model in which isolated segments of the rabbit carotid artery are perfused at different levels of intraluminal pressure.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Experimental Model
Male New Zealand White rabbits (2.1±0.1 kg body wt, n=53) were anesthetized with pentobarbitone sodium (60 mg/kg IV; Sigma-Aldrich) and exsanguinated. The left and right common carotid arteries were dissected, cleansed of adventitial adipose and connective tissue, and cut in half. The 4 segments were mounted into a specially designed 4-position perfusion chamber, where they were stretched back to their in situ length (20.6±0.3 mm, n=152) with the aid of moveable cannulas. Their diameter was continuously monitored by videomicroscopy (Visitron Instruments). The lumen of the segments and the surrounding tissue baths were individually perfused (lumen: 1 mL/min; bath: 0.5 mL/min) with warmed (37°C), oxygenated (lumen: 75% N2, 20% O2, 5% CO2; PO2=140 mm Hg, PCO2=15 to 20 mm Hg, pH 7.4; bath: 95% CO2, 5% O2; PO2 >300 mm Hg, PCO2=18 to 38 mm Hg, pH 7.4) Tyrode solution of the following composition (in mmol/L): Na+ 144.3, K+ 4.0, Cl- 138.6, Ca2+ 1.7, Mg2+ 1.0, HPO42- 0.4, HCO3- 19.9, D-glucose 10.0. An IPC roller pump (Ismatec) was used for perfusion, pumping at a frequency of 1.33 Hz. After a 30-minute equilibration period, the segments were perfused at 2±1, 90±3, or 160±4 mm Hg for 3 to 12 hours with the aid of an adjustable afterload device system (Hugo Sachs Elektronik). Perfusion pressure without the afterload device attached was 2 mm Hg, as determined with a pressure transducer (Gould) connected to a side arm of the outflow tubing.
In experiments with drug or vehicle pretreatment, the segments were perfused with defined drug doses at a reduced flow rate (0.5 mL/min) for up to 1 hour directly after the resting phase. In another series of experiments, the segments were mechanically denuded by gentle abrasion with a roughened stainless steel cannula (2.0 mm OD). At the end of the perfusion, the segments were snap-frozen in liquid nitrogen and stored at -80°C.
Superfusion Bioassay
Superfusion bioassay experiments with 3- to 4-mm-wide ring
segments of the rabbit carotid artery were performed essentially as
described8 except that passive tension was adjusted to
2 g for experiments with ET-1 or sarafotoxin 6c (Alexis).
Staining of Nuclear DNA With Hoechst 33342
After perfusion of the segments with Tyrode solution for 6 hours
at 2 or 160 mm Hg, they were incubated in Waymouth/Ham F12 medium
1:1 (vol/vol) containing 20% fetal bovine serum (Gibco BRL via Life
Technologies) and standard additives for 18 hours. Thereafter, the
segments were fixed with 5% (vol/vol) formaldehyde in 145 mmol/L
sodium chloride, 10 mmol/L HEPES for 12 hours at 4°C followed by
the addition of the DNA-binding fluorescent dye Hoechst
333429 (10 µg/mL; Calbiochem) for 72 hours. The fixed
dye-treated segments were then embedded in paraffin blocks, of which
3-µm-thick sections were cut that were examined in a blinded fashion
by 2 independent investigators with the aid of a videoimaging system
(Visitron Instruments) and the MetaMorph 3.51 software package
(Universal Imaging). The media of 2 sections from each segment were
evaluated at 800-fold magnification, and only those nuclei were judged
apoptotic that showed definite chromatin condensation or
fragmentation. On average, a total of 682 nuclei were counted per
section.
Reverse Transcription–Polymerase Chain Reaction Analysis
The frozen segments were minced under liquid nitrogen with the
aid of a mortar and pestle. Total RNA was isolated with the Qiagen
RNeasy kit (Qiagen) followed by cDNA synthesis with a maximum of 3 µg
RNA and 200 U Superscript II reverse transcriptase (Gibco, Life
Technologies) in a total volume of 20 µL, according to the
manufacturer’s instructions. For normalization of cDNA load, 5 µL
(
75 ng cDNA) of the resulting cDNA solution and 20 pmol of each
primer (Gibco) were used for elongation factor 1 (EF-1) polymerase
chain reaction (PCR) with 1 U Taq DNA polymerase (Gibco) in a total
volume of 50 µL according to the manufacturer’s instructions. PCR
products were electrophoretically separated on 1.5% agarose gels
containing 0.1% ethidium bromide, and the intensity of the detected
bands was determined densitometrically to adjust cDNA volumes for
subsequent PCR analyses.10 All PCR reactions were
performed individually for each primer pair in a Hybaid OmnE
thermocycler (AWG) that was programmed as follows: A unique 2-minute
period for complete denaturation at 94°C in the beginning followed by
a primer-specific number of cycles of 30-second
denaturation at 94°C, 30-second annealing at 53° to 60°C (see
below), and 1-minute primer extension at 72°C, with an additional 5
minutes at 72°C for final extension in the end. To ensure that
the PCR amplification was indeed semiquantitative, several PCR runs
were performed on each set of samples to establish the adequate numbers
of cycles that usually corresponded to those indicated. Individual PCR
conditions were as follows: Prepro ET-1: product size 517 bp, 33
cycles, annealing temperature 53°C,
(forward)5'-GCTCCTGCTCCTCGCTGAT-3', (reverse)
5'-AAGAGCGAGTGAGAGAGTGA-3' (corresponding to
nucleotide sequences 270 to 289 and 786 to 767 of the
rabbit prepro ET-1 gene; GenBank accession No. X59931); ECE-1: 309 bp,
29 cycles, 58°C, 5'-GCACCCTCAAGTGGATGGAC-3',
5'-CCGGAAACACGATCT-CGTTC-3'(1425–1444, 1734–1715, human ECE-1,
Z35307); ETA-R: 334 bp, 30 cycles, 58°C,
5'-CAGGGCATCCTTTTGGCTGGC ACTG-3',
5'-GCGCGTTGGGGCCATTCCTCATAC-3' (24–48, 358–335, human
ETA-R, E07649); ETB-R: 446
bp, 33 cycles, 53°C, 5'-GTGCT-GGGGATCATCGGGAAC-3',
5'-TGAACGGGATGAAGCAAGCAG-3'(570–590, 1015–995, human
ETB-R, E07650) or 304 bp, 33 cycles, 53°C,
5'-TGTTGGCTTCCCCTTCATCT-3',
5'-TGGAGCGGAAGTTGTCGTAT-3'(1203–1219, 1506–1487, rat
ETB)-R, X57764); EF-1: 951 bp, 22 cycles, 58°C,
5'-TGCCGTCCTGATTGTTGCTGC-3',
5'-ATCACGGACAGCGAAACGACC-3' (346–366, 1297–1276, rabbit
EF-1, X62245); CD31 (PECAM-1): 362 bp, 30 cycles, 56°C,
5'-AACTTCACCATCCAGAAGG-3',
5'-CACTGGTATTCCACGTCTT-3'(1207–1225, 1568–1550, human
CD31, M28526); iNOS (inducible nitric oxide synthase): 576 bp, 30
cycles, 60°C, 5'-CAG CTACTGGGTC-AAAGACAAGAGG- 3',
5'-TGCTGAGAGTCATGGAGCCG-3'(543–567, 1118–1099, rabbit
iNOS, U85094).
To verify the identity of the amplification products with the designed primer pairs, we cloned and sequenced the ECE-1, ETA-R, ETB-R, EF-1, CD31, and iNOS PCR products and found an 87% to 92% homology with the published sequences of the corresponding human and rat genes. The homology of the ppET-1 PCR product with the corresponding rabbit gene was 100%.
Measurements of ET-1 Tissue Concentrations
ET-1 was extracted from the weighted segments according to the
methods of Hisaki et al11 and Moreau et al,12
with minor modifications. Overall recovery of ET-1 was 69.4%;
interassay and intra-assay variabilities were 15.3% and 11.7%,
respectively. The concentration of ET-1 in the tissue extracts was
determined with the use of a commercially available ELISA kit
(Amersham) according to the manufacturer’s instructions.
Electrophoretic Mobility Shift Analysis
Preparation of nuclear extracts from the minced segments,
incubations with 10 µg nuclear protein and 10 to 20 000 cpm of the
double-stranded consensus oligodeoxynucleotides (Santa Cruz
Biotechnology), nondenaturing polyacrylamide gel (4%)
electrophoresis, autoradiography, and supershift
analyses were performed essentially as
described.10 The single-strand sequences of the
oligodeoxynucleotides were as follows (core sequences are
italicized): Activator protein-1 (AP-1),
5'-CGCTTGATGACTCA-GCCGGAA-3'; CCAAT enhancer binding
protein (C/EBP), 5'-TGCAGATTGCGCAATCTGCA-3';
nuclear factor-
B (NF-B), 5'-AGTTGAGGGGACTTTCCCAGG-3';
CREB, 5'-AGAGATTGCC-TGACGTCAAGAAGCTAG-3'; GATA,
5'-CACTTGATAACAGA-AAGTGATAACTCT-3'; egr-1,
5'-GGATCCAGCGGGGGCGA-GCGGGGGCGA-3'; ets-1,
5'-GATCTCGAGCAGGAAGTTCGA-3'; Sp1,
5'-ATTCGATCGGGGCGGGGCGAGC-3'.
Statistical Analysis
Unless indicated otherwise, all data in the figures and text are
expressed as mean±SEM of n observations. Statistical evaluation was
performed by Student’s t test for unpaired data with the
Instat for Windows statistics software package (GraphPad Software Inc),
and a value of P<0.05 was considered statistically
significant.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Pressure-Diameter Relation
Raising the intraluminal pressure from 2 to 220 mm Hg resulted in a progressive increase in (outer) diameter of the perfused segments that reached a maximum at 160 mm Hg (Figure 1a), with an average distention of 208±6% (n=76). Of note was that the segments were already distended close to the maximum at the physiological pressure of 90 mm Hg. Circumferential (tensile) strain (
), defined as
pressure-induced change in diameter (d-d0) as a
fraction of the diameter at zero pressure
(d0),13 14 was also maximal at
160 mm Hg and close to the maximum at 90 mm Hg (compare
with Figure 1a).
|
Control Experiments
The mRNA level of the housekeeping reference gene, EF-1, was not
altered by an increase in perfusion pressure under any of the
experimental conditions described below (compare with Figure 1b). Moreover, there was no apparent loss of EC from the
endothelium-intact perfused segments, even after 12
hours of exposure to 160 mm Hg, as judged by the virtually
constant expression of the EC-specific marker CD31 (Figure 1c)
and light microscopy analysis of paraffin-embedded hematoxylin
and eosin–stained tissue sections as well as transmission electron
microscopy (not shown). Even though the experiments were not performed
under sterile conditions, there was no microscopically visible
contamination at any time point and no expression of iNOS mRNA, an
extremely sensitive marker for the presence of bacterial
lipopolysaccharides (not shown).
Prepro ET-1 Expression and ET-1 Synthesis
In endothelium-intact segments, a distinct basal
expression of ppET-1 was detectable that decreased by 61±13% (n=4,
P<0.05) after mechanical denudation. Efficient EC removal
was verified by a 93% loss of CD31 mRNA abundance in the denuded
segments (Figure 1d). Perfusion at either 2 or 90 mm Hg
for up to 9 hours had no appreciable effect on ppET-1 mRNA abundance in
endothelium-intact segments (Figure 2a), which, on the other hand, was
markedly elevated when the segments were perfused at 160 mm Hg
for 3 to 12 hours (Figures 2a and 2b). As shown in Figure 2b, this pressure-induced increase in ppET-1 mRNA was confined
to the EC. Moreover, a comparable pressure-induced increase in ppET-1
mRNA was observed when the segments were first equilibrated at 90
mm Hg for 3 hours and then exposed to 160 mm Hg for 6 hours
(Figure 2a). For reasons of simplicity,
therefore, all other experiments were performed by comparing segments
perfused at 2 mm Hg with those perfused at 160 mm Hg. In
addition to the increase in ppET-1 mRNA (917%), the intravascular
concentration of ET-1 peptide was elevated to a similar extent (931%)
after 6-hour perfusion at 160 mm Hg (Figure 4a).
|
|
P<0.05 vs
160 mm Hg.
ECE-1 and ETA-R Expression
In contrast to ppET-1, there was no pressure-induced increase in
ECE-1 mRNA under any of the aforementioned experimental conditions
(compare with Figure 2, a and b). ETA-R
mRNA expression was also largely unaffected by raising the intraluminal
pressure to 160 mm Hg for 3 to 12 hours (compare with Figure 3b for the lack of effect after 6 hours), although in some
experiments a small increase was noted (Figure 3a).
|
ETB-R Expression
ETB-R mRNA abundance, on the other hand, was
significantly increased after 3 to 12 hours of exposure to 160
mm Hg (inserts in Figures 2a, 3a, and 3b). This
pressure-induced increase in ETB-R mRNA, however,
was reproducibly detectable only in the rostral part, that is, next to
the bifurcation of the internal and external carotid artery (Figure 3c). Both basal and pressure-induced ETB-R
expression appeared to be largely confined to the SMC (Figure 3b
insert). Parallel analysis of 2 completely different PCR
products, designed from the human and rat
ETB-R sequence, corroborated this
pressure-induced rise in ETB-R mRNA abundance
(341±24% and 342±55% of 2 mm Hg, respectively, n=6). Raising
the intraluminal pressure further (ie, to 250 mm Hg) finally
resulted in an increase in ETB-R mRNA also in the
caudal segments (218% as compared with 506% of control at 160
mm Hg for the rostral segment from the same animal). The different
pressure sensitivity of ETB-R expression did not
appear to be due to a greater dilatability of the rostral (=0.93,
n=39) as compared with the caudal segments (=1.14, n=37). Unlike
ETB-R expression, there was no difference in the
pressure-induced rise in ppET-1 mRNA abundance between the 2
parts of the carotid artery (Figure 3c).
Because both Western blot and [125I]-ET-1 receptor binding analyses failed for technical reasons to confirm that in addition to the pressure-induced increase in ETB-R mRNA there is a corresponding increase in ETB-R protein, the superfusion bioassay technique was used. However, despite being sensitive toward ET-1 (threshold 30 pmol), no constrictor response to the specific ETB-R agonist15 16 sarafotoxin 6c, up to a dose of 1 nmol (corresponding to a final concentration of 1 µmol/L), was observed (not shown). The ET-1–induced constriction, on the other hand, was markedly reduced (to 14±3% of control at a concentration of 0.1 µmol/L ET-1, n=3, P<0.05) by the selective ETA-R antagonist4 BQ 123 (1 µmol/L).
Therefore, another assay method had to be established that finally demonstrated a pressure-dependent increase in functional ETB-R protein. Preliminary findings from this laboratory had indicated that ET-1 through activation of the ETB-R exerts a proapoptotic effect on rat aortic cultured smooth muscle cells (M. Cattaruzza, C. Dimigen, and M. Hecker, unpublished data, 1999). In the media of endothelium-intact segments of the rabbit carotid artery (rostral part), an increased number of apoptotic nuclei, characterized by chromatin condensation and fragmentation, was detected 18 hours after the segments had been exposed to a perfusion pressure of 160 mm Hg for 6 hours. Moreover, this pressure-induced increase in apoptosis was completely prevented by the ETB-R–selective antagonist4 BQ 788 but not by BQ 123 (1 µmol/L each; Figure 4b). This increase in chromatin condensation was preceded by a rise in caspase-3 activity in homogenates of the segments (not shown), which in combination justify the term apoptosis.
Transcriptional Control of Pressure-Induced Gene Expression, Role
of Protein Kinases
Blockade of RNA synthesis with actinomycin D (1 µmol/L)
abolished the pressure-induced increase in ppET-1 and
ETB-R mRNA (Figure 5a) as well as the increase in
intravascular ET-1 (Figure 5a, insert). Basal ppET-1 and
ETB-R mRNA but not ET-1 peptide levels (Figure 5a, insert) were also reduced by actinomycin D.
|
Pretreatment of the segments with the protein kinase C (PKC) inhibitor17 Ro 31-8220 (0.1 µmol/L) or the c-Src family–specific tyrosine kinase inhibitor18 herbimycin A (0.1 µmol/L) for 1 hour had no significant effect on ECE-1 or ETA-R expression both under basal conditions and in the presence of an elevated perfusion pressure (not shown). In contrast, Ro 31-8220 significantly attenuated the ETB-R mRNA level after 6-hour perfusion both at 2 mm Hg (to 43±15% of control, n=5, P<0.05) and 160 mm Hg (Figure 5b). In contrast, Ro 31-8220 did not affect basal and only marginally inhibited the pressure-dependent expression of ppET-1 (Figure 5b). Herbimycin A, on the other hand, abolished the pressure-induced increase in ppET-1 mRNA but had no effect on ETB-R mRNA abundance both under basal conditions and after 6-hour exposure to 160 mm Hg (Figure 5c).
Among several transcription factors that are potentially involved in
the control of ppET-1 and ETB-R expression
(analysis of the 5'-flanking region of the rat ppET-1[GenBank
accession No. S76970] and human ETB-R gene
[GenBank accession No. D13162] with the use of the Matinspector V2.2
software package19 ), only activator protein-1
(AP-1) and 2 members of the C/EBP family of transcription factors, ß
and (according to supershift analysis, not shown), revealed
a prominent translocation to the nucleus when perfusion pressure was
raised from 2 to 160 mm Hg for up to 2 hours (Figure 6). In contrast, there was no appreciable
difference in CREB, egr-1, ets-1, GATA, NF-B, or Sp1 activity
between pressurized and nonpressurized segments over this period of
time (not shown). Activation of AP-1 and C/EBP was transient with a
nadir at 30 to 60 minutes, and there was no difference in the pressure
sensitivity of both transcription factors between rostral and caudal
segments. Both basal and pressure-induced AP-1 activity was strongly
inhibited by Ro 31-8220 (Figure 7a),
whereas the PKC inhibitor had no significant effect on the
activity of C/EBP (not shown). Herbimycin A, on the other hand, had no
significant effect on AP-1 activity (not shown) but completely
abrogated the pressure-induced activation of C/EBP (Figure 7b).
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Although the chosen experimental conditions do not precisely match the situation in the rabbit carotid artery in vivo, the newly developed model allowed us to study the effect of a pathophysiological increase in intraluminal pressure on gene expression in the vessel wall in situ. With the aid of this model, we were able to substantiate earlier findings in cultured EC of an increase in ppET-1 expression6 and ET-1 release5 in response to a presumably undirected deformation of these cells. This deformation-induced gene expression did not follow the pattern of an all-or-nothing response but was clearly dependent both on the duration and intensity of the increase in perfusion pressure. Of note was that it occurred only at supra-physiological pressure levels (>90 mm Hg), whereas circumferential (tensile) strain at 90 mm Hg was nearly maximal. This suggests that in addition to circumferential strain, another hemodynamic force (ie, radial strain or the stiffening of the vessel wall when collagen takes over from elastin to balance the distending pressure)13 14 must contribute to the deformation of the cells in the vessel wall. This hypothesis is substantiated by our finding of an additional pressure-induced increase in smooth muscle ETB-R expression only in the rostral part of the rabbit carotid artery, even though the dilatability of the caudal segments was very similar. Moreover, the finding of a virtually indistinguishable pressure-dependent increase in ppET-1 expression in both parts of the carotid artery suggests that certain thresholds exist for deformation-induced gene expression in the vessel wall and that this threshold is lower for ppET-1 than for ETB-R expression. Alternatively, it may be argued that EC are either more easily deformed than SMC or deformed differently with other components of the cytoskeleton being affected. In contrast to ppET-1 and the ETB-R, neither expression of the ETA-R nor that of ECE-1 appeared to be pressure-sensitive per se.
Because the deformation-induced increase both in ppET-1 and ETB-R mRNA and ET-1 peptide content was sensitive to actinomycin D, it would appear that it is controlled at the level of mRNA synthesis. In a first attempt to elucidate the signal transduction pathway involved therein, Ro 31-8220, a potent and highly selective inhibitor of various PKC isoforms,17 was used. PKC blockade, however, did not affect the pressure-induced expression of ppET-1 in the endothelium, whereas smooth muscle ETB-R expression was completely inhibited. Pressure-induced ppET-1 but not ETB-R expression was abolished, on the other hand, by the c-Src family tyrosine kinase inhibitor18 herbimycin A. It would appear, therefore, that the pressure-dependent deformation of the carotid artery wall is sensed differently by EC and SMC, involving activation of the c-Src and protein kinase C pathways, respectively.20
Moreover, the differential inhibition by Ro 31-8220 and herbimycin A of
the activation of AP-1 and C/EBP suggests that C/EBP (ß and/or )
is an essential transcription factor for the pressure-induced
expression of ppET-1 in the venous EC, whereas AP-1 is involved in the
pressure-dependent expression of the ETB-R in the
SMC. Hitherto, a cooperative interaction of GATA-2 and AP-1 is thought
to regulate transcription of the ppET-1 gene,21 whereas a
functional role for C/EBP in ppET-1 gene expression has not yet been
recognized. The transcriptional control of ETB-R
expression has not been investigated thus far. Our data, therefore,
provide the first indirect evidence for a role of AP-1 in
ETB-R expression.
In addition to providing a basis for a more detailed investigation of the nuclear signaling mechanisms involved in hypertension-induced gene expression in the vessel wall in situ, the aforementioned findings raise the question as to what the functional consequences of the pressure increase in ET-1 synthesis and ETB-R expression are. With the superfusion bioassay technique, no ETB-R–mediated vasoconstriction could be detected (compare with References 15 and 1615 16 ). On the other hand, we noted a pressure-dependent ETB-R–mediated increase in apoptosis of the medial SMC, a finding that clearly points to a functional role of the ETB-R in the rabbit carotid artery other than influencing SMC tone, as does the ETA-R. In this context, it was interesting to note that arterial SMC have been reported to undergo apoptosis in experimental hypertension and that this response is aggravated by ETA-R blockade.22 Provided that the observed enhanced rate of apoptosis represents a general phenotypic change of the SMC and is transferable to the situation in human blood vessels, it may ultimately lead to the media hypertrophy that is characteristic for hypertension-induced remodeling in conduit arteries.
In summary, the aforementioned findings reinforce the notion of a hypertension-induced rise in ET-1 synthesis in the endothelium along with an increased expression of the ETB-R in the smooth muscle of large conduit arteries. These blood pressure–induced changes in gene expression may contribute to arterial remodeling in hypertension, hence promoting the manifestation of this disease.
|
Acknowledgments |
|---|
This work was supported by the Deutsche Forschungsgemeinschaft (He 1587/5-2 and 7-1). The authors are indebted to Drs Andreas Wagner and Ekkehard Schütz (Department of Clinical Chemistry, University of Goettingen) for their help with the electrophoretic mobility shift analysis and the sequencing of the PCR amplification products, respectively.
Received September 2, 1999; first decision September 24, 1999; accepted October 6, 1999.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
- Langille BL. Arterial remodeling: relation to hemodynamics. Can J Physiol Pharmacol. 1996;874:834–841.
- Schiffrin, EL, Intengan HD, Thibault G, Touyz RM. Clinical significance of endothelin in cardiovascular disease. Curr Opin Cardiol. 1997;12:354–367.[Medline] [Order article via Infotrieve]
- Parris RJ, Webb DJ. The endothelin system in cardiovascular physiology and pathophysiology. Vasc Med. 1997;2:31–43.[Medline] [Order article via Infotrieve]
- Douglas SA, Ohlstein EH. Signal transduction mechanisms mediating the vascular actions of endothelin. J Vasc Res. 1997;34:152–164.[Medline] [Order article via Infotrieve]
- Sumpio BE, Widmann MD. Enhanced production of endothelium-derived contracting factor by endothelial cells subjected to pulsatile stretch. Surgery. 1990;108:277–281.[Medline] [Order article via Infotrieve]
- Wang DL, Wung BS, Peng YC, Wang JJ. Mechanical strain increases endothelin-1 gene expression via protein kinase C pathway in human endothelial cells. J Cell Physiol. 1995;163:400–406.[Medline] [Order article via Infotrieve]
-
Davies PF. Flow-mediated endothelial
mechanotransduction. Physiol Rev. 1995;75:519–560.
[Abstract/Free Full Text] - Kaw S, Hecker M. Endothelium-derived hyperpolarizing factor, but not nitric oxide or prostacyclin release, is resistant to menadione-induced oxidative stress in the bovine coronary artery. Naunyn Schmiedeberg’s Arch Pharmacol. 1999;359:133–139.[Medline] [Order article via Infotrieve]
- Surh CD, Kishimoto H, Sprent J. Morphological, biochemical and flow cytometric assays of apoptosis. In: Coligan JE, Kruisbeek AM, Margulies DH, Shevach EM, Strober W, eds. Current Protocols in Immunology, unit 3.17. New York, NY: John Wiley & Sons; 1999.
-
Krzesz R, Wagner AH, Cattaruzza M, Hecker M.
Cytokine-inducible CD40 gene expression in vascular smooth
muscle cells is mediated by nuclear factor B and signal
transducer and activator of transcription-1. FEBS
Lett. 1999;453:191–196.[Medline]
[Order article via Infotrieve]
-
Hisaki K, Matsumura Y, Maekawa H, Fujita K, Takaoka M,
Morimoto S. Conversion of big ET-1 in the rat lung: role of
phosphoramidon-sensitive endothelin-1-converting
enzyme. Am J Physiol. 1994;266:H422–H428.
[Abstract/Free Full Text] -
Moreau P, d’Uscio LV, Shaw S, Takase H, Barton M,
Lüscher TF. Angiotensin II increases tissue
endothelin and induces vascular hypertrophy.
Circulation. 1997;96:1593–1597.
[Abstract/Free Full Text] - Dobrin Ph. Vascular mechanics. In: Shepherd JT, Abboud FM, eds. Handbook of Physiology. Section 2: The Cardiovascular System, Vol 3: Peripheral Circulation and Organ Blood Flow, Part I. Bethesda, Md: American Physiological Society; 1983:65–102.
- Caro CG, Pedley TJ, Schroter RC, Seed WA. The Mechanics of the Circulation. Oxford, UK: Oxford University Press; 1978.
- White DG, Cannon TR, Garratt H, Mundin JW, Sumner MJ, Watts IS. Endothelin ETA and ETB receptors mediate vascular smooth-muscle contraction. J Cardiovasc Pharmacol. 1993;22:S144–S14.
- Lodge NJ, Halaka NN. Endothelin receptor subtype(s) in rabbit jugular vein smooth muscle. J Cardiovasc Pharmacol. 1993;22:S140–S143.
- Wilkinson SE, Parker PJ, Nixon JS. Isoenzyme specificity of bisindolylmaleimides, selective inhibitors of protein kinase C. Biochem J. 1993;294:335–337.
- Takahashi M, Berk BC. Mitogen-activated protein kinase (ERK1/2) activation by shear stress and adhesion in endothelial cells: essential role for a herbimycin-sensitive kinase. J Clin Invest. 1996;98:2623–2631.[Medline] [Order article via Infotrieve]
-
Quandt K, Frech K, Karas H, Wingender E, Werner T.
MatInd, and MatInspector: new fast and versatile tools for detection of
consensus matches in nucleotide sequence data.
Nucleic Acids Res. 1995;23:4878–4884.
[Abstract/Free Full Text] -
Lehoux S, Tedgui A. Signal transduction of mechanical
stresses in the vascular wall. Hypertension. 1998;32:338–345.
[Abstract/Free Full Text] - Kawana M, Lee ME, Quertermous EE, Quertermous T. Cooperative interaction of GATA-2 and AP-1 regulates transcription of the endothelin-1 gene. Mol Cell Biol. 1995;15:4225–4231.[Abstract]
- Sharifi AM, Schiffrin EL. Apoptosis in aorta of deoxycorticosterone acetate-salt hypertensive rats: effect of endothelin receptor antagonism. J Hypertens. 1997;15:1441–1448.[Medline] [Order article via Infotrieve]
This article has been cited by other articles:
 |
|
 |
|
  D. G. Sedding, M. Homann, U. Seay, H. Tillmanns, K. T. Preissner, and R. C. Braun-Dullaeus Calpain counteracts mechanosensitive apoptosis of vascular smooth muscle cells in vitro and in vivo FASEB J, February 1, 2008; 22(2): 579 - 589. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Wendel, W. Kummer, L. Knels, J. Schmeck, and T. Koch Muscular ETB Receptors Develop Postnatally and Are Differentially Distributed in Specific Segments of the Rat Vasculature J. Histochem. Cytochem., February 1, 2005; 53(2): 187 - 196. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Gschwend, R. H. Henning, D. de Zeeuw, and H. Buikema Coronary Myogenic Constriction Antagonizes EDHF-Mediated Dilation: Role of KCa Channels Hypertension, April 1, 2003; 41(4): 912 - 918. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. H.S. Kim and L. J. Rubin Endothelin in Health and Disease: Endothelin Receptor Antagonists in the Management of Pulmonary Artery Hypertension Journal of Cardiovascular Pharmacology and Therapeutics, March 1, 2002; 7(1): 9 - 19. [Abstract] [PDF]
|
|
|
|
 |
|
 A. B. Buchwald, A. H. Wagner, C. Webel, and M. Hecker Decoy oligodeoxynucleotide againstactivator protein-1 reducesneointimal proliferation after coronaryangioplasty in hypercholesterolemic minipigs J. Am. Coll. Cardiol., February 20, 2002; 39(4): 732 - 738. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. S. Filippatos, N. Gangopadhyay, O. Lalude, N. Parameswaran, S. I. Said, W. Spielman, and B. D. Uhal Regulation of apoptosis by vasoactive peptides Am J Physiol Lung Cell Mol Physiol, October 1, 2001; 281(4): L749 - L761. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. H. Wagner, R. Krzesz, D. Gao, C. Schroeder, M. Cattaruzza, and M. Hecker Decoy Oligodeoxynucleotide Characterization of Transcription Factors Controlling Endothelin-B Receptor Expression in Vascular Smooth Muscle Cells Mol. Pharmacol., April 13, 2001; 58(6): 1333 - 1340. [Abstract] [Full Text]
|
|
|
|
 |
|
 M. Lauth, M. Cattaruzza, and M. Hecker ACE Inhibitor and AT1 Antagonist Blockade of Deformation-Induced Gene Expression in the Rabbit Jugular Vein Through B2 Receptor Activation Arterioscler. Thromb. Vasc. Biol., January 1, 2001; 21(1): 61 - 66. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Walsh, R. C. Smith, and H.-S. Kim Vascular Cell Apoptosis in Remodeling, Restenosis, and Plaque Rupture Circ. Res., August 4, 2000; 87(3): 184 - 188. [Full Text] [PDF]
|
|
|
|
|
|
M. CATTARUZZA, C. DIMIGEN, H. EHRENREICH, and M. HECKER Stretch-induced endothelin B receptor-mediated apoptosis in vascular smooth muscle cells FASEB J, May 1, 2000; 14(7): 991 - 998. [Abstract] [Full Text]
|
|
|
|
 |
|
 M. Cattaruzza, I. Eberhardt, and M. Hecker Mechanosensitive Transcription Factors Involved in Endothelin B Receptor Expression J. Biol. Chem., September 28, 2001; 276(40): 36999 - 37003. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||