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Department of Cardiovascular Physiology, University of Goettingen; and
* Max-Planck-Institute for Experimental Medicine and Departments of Neurology and Psychiatry, University of Goettingen, Germany
1Correspondence: Department of Cardiovascular Physiology, University of Goettingen, Humboldtallee 23, 37073 Goettingen, Germany. E-mail hecker{at}veg-physiol.med.uni-goettingen.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: vascular remodeling • pressure overload • endothelin system • hypertrophy
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Both effects appear to be mediated through activation
of the G-protein-coupled type A receptor (ETA-R;
2
), predominantly localized on these cells (3
, 4)
. As the synthesis of ET-1 was reported to be increased in
several hypertrophic and/or hyperplastic vascular diseases (5
, 6)
, it has been implicated in the adaptive response of VSMC to
an aberrant mechanical stress such as the chronic pressure overload in
arterial hypertension (7
; for review, see ref
8
).
In contrast to the ETA-R, the
ETB-R is expressed both on endothelial cells and
VSMC. Its activation promotes a G-protein-mediated increase in
intracellular Ca2+ similar to that of the
ETA-R (9)
. Its functional
significance, however, is still ill-defined. Thus, activation of the
ETB-R is responsible for the transient
endothelium-mediated depressor response to ET-1 in vivo; on
the other hand, it also promotes venoconstriction in most vascular
beds. The ETB-R-mediated constriction in arteries
is usually not detectable, however, or is rather weak (10
, 11)
.
As with their relative functional significance, regulation of
expression of the endothelin receptors is still poorly understood, but
hemodynamic factors such as shear stress and wall tension may well
influence their abundance in vivo, as is the case with
prepro-ET-1 (12
, 13)
.
Therefore, we set up an in vitro model to study the influence of cyclic strain on the expression of the endothelin system in vascular cells. Moreover, we investigated possible consequences of such a deformation-induced change in gene expression.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell culture
Smooth muscle cells (raSMC) from the aorta of male Wistar rats
(200–250 g body weight) were isolated by the explant technique and
cultured in 1 ml of Waymouth medium supplemented with 10% fetal bovine
serum (FBS), 50 U/ml penicillin, 50 µg/ml streptomycin, 10 U/ml
nystatin, 5 mM HEPES, and 5 mM TES, as described previously
(14)
.
RaSMC at passage 2 or 3 were seeded into Bioflex collagen type 1 coated
6-well plates (Flexcell Inc., Hillsborough, N.C.) and used for
experiments at ~70% confluence. Their identity was confirmed after
fixation with p-formaldehyde by positive immunostaining for
smooth muscle 
-actin with a monoclonal anti- smooth muscle actin
antibody and a secondary anti-mouse IgG-FITC conjugate from goat
(Sigma-Aldrich). According to this procedure, the cultured smooth
muscle cells appeared to be essentially homogeneous. RaSMC derived from
wild-type (+/+), heterozygous (sl/+), and homozygous (sl/sl) spotting
lethal rats (10–50 g body weight) were prepared and cultured
essentially as described above. Breeding of the heterozygous animals
and genotyping by reverse transcription-polymerase chain reaction
(RT-PCR) analysis were performed as described previously
(15)
. All experiments with raSMC from these animals were
conducted in a comparative fashion (i.e., each set of experiments was
carried out with cells isolated from littermates).
Endothelial cells (PAEC) were isolated from porcine aortas by treatment
with 1.2 U/ml dispase (Roche Diagnostics, Mannheim, Germany) in
HEPES-modified Tyrode solution (total volume of 3 ml per aorta) for 7
min at 37°C. They were cultured on BioFlex collagen type I 6-well
plates that had also been coated with gelatin (2 mg/ml gelatin in 0.1 M
HCl for 30 min at ambient temperature) in DMEM-Ham’s F12 (1:1, v/v,
Life Technologies) containing 10 U/ml nystatin, 50 U/ml penicillin, 50
µg/ml streptomycin, 5 mM HEPES, 5 mM TES, and 20% FBS until they
reached confluence. They were identified by their typical cobblestone
morphology and after fixation with p-formaldehyde by
positive immunostaining for von Willebrand factor (vWF), with a
polyclonal rabbit anti-vWF serum and a secondary anti-rabbit IgG Texas
red conjugate from sheep (Sigma-Aldrich), and negative immunostaining
for -smooth muscle actin, as described before.
Administration of cyclic strain to cultured cells
If not indicated otherwise, the following standard protocol was
performed. The conditioned medium of the cultured cells was exchanged
against 2 ml of fresh medium supplemented as described, except for the
omission of serum, 2 h before the start of the experiment. If any
test compounds were to be administered, they were added 30 min earlier.
After this period, the plates were mounted in a FlexerCell FX-3000
strain unit (Flexcell) placed in an incubator. RaSMC were stretched for
6 h with up to 20% elongation at 0.5 Hz. Elongation time was
adjusted to 50% of total running time, resulting in cycles of 1 s
of stretch and 1 s release, respectively. For PAEC, the program
performed was identical except for the strain rate, which was adjusted
to 12%.
Semi-quantitative RT-PCR analysis
Total RNA was isolated according to the method described by
Chomczynski and Sacchi (16)
. First strand cDNA synthesis
from ~3 µg of total RNA was performed with Superscript reverse
transcriptase (Life Technologies) according to the manufacturer’s
instructions. To normalize cDNA amounts in the samples from one
experiment, 2.5% of the resulting cDNA was used for performing PCR
reactions for the housekeeping gene, elongation factor 2 (EF-2). PCR
was performed with as few cycles as possible to clearly detect the PCR
products on an ethidium bromide-stained agarose gel. According to
densitometric analysis (One-Dscan Gel analysis software from
Scanalytics, Billerica, Mass.) of the PCR products, cDNA volumes were
adjusted for consecutive analyses. Programs and primers for the
measurement of steady-state levels of mRNA of EF-2 and the other gene
products were as follows: EF-2for: 5'-GAC ATC ACC AAG GGT GTG CAG-3';
EF-2rev: 5'-GCG GTC AGC ACA CTG GCA TA-3' (218 bp, position 1990 to
2207 of the human cDNA sequence); ECE-1for: 5'-CGT AGC GAT AGT CTT AGC
AC-3'; ECE-1rev: 5'-GTG CCA CAC CAA AAC TAC AG-3' (529 bp, position
3815 to 4324, rat cDNA sequence); ET-1for: 5'-GGA GCT CCA GAA ACA GCT
GTC-3'; ET-1rev: 5'-CTG CTG ATA AAT ACA CTT CTT TCC-3' (432 bp,
position 233 to 664, rat cDNA sequence); ETAfor:
5'-TTC GTC ATG GTA CCC TTC GA-3'; ETArev: 5'-GAT
ACT CGT TCC ATT CAT GG -3' (546 bp, position 713 to 1258, rat cDNA
sequence); ETBfor: 5'-TTC ACC TCA GCA GGA TTC
TG-3'; ETBrev: 5'-AGG TGT GGA AAG TTA GAA CG-3'
(475 bp, position 1216 to 1670, rat cDNA sequence).
All PCR reactions were performed in OmnE thermocyclers from Hybaid
(Heidelberg, Germany). The primers for EF-2 were kindly provided by Dr.
E. Schütz, Department of Clinical Chemistry, University of
Goettingen. Primers for the endothelin receptors were from Wang et al.
(5)
. For all primers, 58°C was established to be the
optimal annealing temperature. The program performed for PCR
amplification included an initial period of 2 min at 94°C, followed
by a variable number of cycles of 30 s denaturation at 94°C,
30 s annealing at 58°C, and finally 60 s extension at
72°C. The program was terminated with a period of 5 min at 72°C. To
be within the exponential phase of the semi-quantitative PCR reaction,
the appropriate number of cycles (i. e. the phase of PCR when
approximately a doubling of product was achieved with every cycle) was
newly established for every set of samples.
Determination of ET-1 release
After 6–12 h of incubation, the conditioned medium of the
cultured cells was collected and analyzed for its ET-1 peptide content
with an ELISA kit from Amersham (Pharmacia, Freiburg, Germany)
according to the manufacturer’s instructions.
Caspase-3 assay
RaSMC were harvested with a cell scraper, washed twice in
Hank’s balanced salt solution (HBSS), and resuspended in 2 vol of
lysis buffer (50 mM HEPESxKOH, 50 mM KCl, 5 mM EGTA, 2 mM
MgCl2, 1 mM DTT, pH 7.0). Three cycles of
freeze-thawing in liquid nitrogen and at 37°C in a water bath ensued,
followed by extensive mixing; thereafter, the lysate was sedimented at
4°C and 1500 g for 5 min in an Eppendorf benchtop
centrifuge. The supernatant was diluted 1:5 in ICE buffer [(100 mM
HEPESxKOH, 10% (w/v) sucrose, 0.1% (w/v) Triton X-100, 10 mM DTT and
0.1 mg/ml bovine serum albumin, pH 7.5)] and the protein content was
measured with a modified Bradford protein assay (Bio-Rad, Munich,
Germany) according to the manufacturer’s instructions. The
concentration of protein was adjusted to ~1 mg/ml, and 200 µl of
this extract was incubated for 30 min at 30°C with 4 µl of a 1 mM
solution of the caspase-3 specific substrate, DEVD-AFC
(Asp-Glu-Val-Asp-7-amino-4-trifluoro-methyl-coumarin), yielding a final
substrate concentration of 20 µM. After the incubation, 150 µl of
each sample were transferred into a 96-well plate and the ensuing
fluorescence monitored at 405 nm excitation and 510 nm emission
wavelength. The fluorescence intensity of released AFC was calculated
as nmoles of substrate released per milligram of protein/min by using
standards of known concentration and the SOFTmax
PRO-f software from Molecular Devices (Munich, Germany).
Flow cytometry
RaSMC were washed twice with HBSS and then incubated with 0.5%
trypsin/0.2%EDTA (w/v, Life Technologies) for 10 min at 37°C. After
complete detachment of the cells, the suspension was transferred into a
centrifuge vial, 300 µl of FBS were added to inhibit the trypsin
activity, and the cells were sedimented for 5 min at 4°C and 800
g. The sedimented cells were fixed with 70% ethanol and
incubated for 15 min at ambient temperature. Fixed cells were harvested
by centrifugation at 800 g at ambient temperature and
finally resuspended in HBSS containing 200 µg/ml RNase A
(Sigma-Aldrich) and 50 µg/ml of the DNA-intercalating dye propidium
iodide (Sigma-Aldrich). After incubation for at least 30 min at ambient
temperature, the DNA content per nucleus was analyzed by a FACS Scan
flow cytometer (Becton Dickinson Co., Mansfield, Mass.) as described by
Okazawa et al. (17)
.
Staining of nuclear DNA with H 33342
RaSMC grown on Bioflex membranes were incubated with fixation
buffer (5% formaldehyde in 145 mM NaCl, 10 mM HEPESxKOH, pH 7.5) for
20 min at room temperature. After this period the fluorescent
bisbenzimide dye H 33342 (Calbiochem, Bad Soden, Germany) was added at
a final concentration of 10 µg/ml and the cells were incubated for a
second period of 20 min. Thereafter, the buffer was discarded,
membranes were cut out with a scalpel, and mounted upside down on a
microscope slide with 10 µl of mounting buffer (50% glycerol in
Hank’s balanced salt solution). The slides were examined by using a
video imaging system (Visitron, München, Germany). Nuclear
staining intensity and morphology were evaluated optically and
documented photographically.
Statistical analysis
Unless indicated otherwise, results are expressed as means ± SE of n observations with cells obtained from
the aortas of different animals. Statistical evaluation was performed
by Student’s t test for unpaired data, with a P
value <0.05 considered statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The latter effect occurred at various levels of stretch
(8–20%) and was completely reversible within 18 h after exposure
of the cells to cyclic strain (not shown). At the peptide level, the
amount of ET-1 released from raSMC in static culture amounted to about
a quarter of that released by PAEC and was further decreased on
exposure to cyclic strain, whereas the release of ET-1 from PAEC was
significantly increased, albeit not to the same extent as the prepro
ET-1 mRNA level (Fig. 1A
).
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Differential regulation of ETA-R and ETB-R
expression in stretched raSMC
At the mRNA level, the ETA-R appeared to be
the predominant receptor expressed in raSMC in static culture
(Fig. 2
). Exposure to cyclic strain induced a distinct decrease in
ETA-R mRNA abundance (Fig. 2)
, which lasted up to
6 h after termination of cyclic strain but returned to prestrain
levels within 12 h (not shown). In contrast,
ETB-R mRNA abundance was strongly up-regulated on
exposure to cyclic strain (Fig. 2)
, and this effect was maintained for
at least 12 h after terminating the stretch protocol (not shown).
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ET-1-induced ETB-R-mediated apoptosis in stretched
raSMC
To analyze the functional consequences of this stretch-induced
coordinated regulation of the endothelin system at the level of
transcription, we performed experiments where exogenous ET-1 (10 nM)
was added to the stretched raSMC, thus mimicking the in situ
situation with the endothelium having released a greater amount of ET-1
under these conditions. As judged by FACS analysis 18 h after
termination of the stretch protocol, no significant alterations in
total cell number or relative amounts of cells in S or
G2 phase were observed in the absence of
exogenous ET-1 when comparing the stretched cells to the static
controls (not shown). In contrast, a prominent
sub-G1 peak indicating high amounts of dying
cells was detected in raSMC exposed to both cyclic strain and exogenous
ET-1, but not in cells exposed individually to these stimuli (cf.
Fig. 3
for the lack of effect of stretch alone). This prominent ET-1-mediated
cell death was prevented by BQ 788 (Fig. 3)
but not by BQ 123 (not
shown) at a concentration of 0.1 µM.
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To test the hypothesis of an ET-1-triggered apoptosis in stretched
raSMC, caspase-3 activity (8 h after terminating the stretch protocol,
Fig. 4
) and DNA ladder formation (12–24 h after stretch termination, not
shown) were analyzed. Moreover, chromatin condensation and nuclear
morphology were evaluated by staining with the fluorescent DNA binding
dye, H 33342 (Fig. 5
). All four markers of apoptosis indicated that exogenous ET-1 indeed
induced an ETB-R-mediated apoptosis in raSMC when
the cells had been exposed to cyclic strain before, whereas either
stretch or exogenous ET-1 alone did not alter the basal rate of
apoptosis of these cells(112±18 and 109±15% of caspase 3 activity in
static control cells, respectively, n=4).
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Moreover, administration of BQ 788 alone afforded a protective effect on the stretched cells (30% decrease in the number of apoptotic nuclei in three of four experiments), whereas BQ 123 alone tended to enhance the rate of apoptosis (approximate doubling of the number of apoptotic nuclei in two of four experiments).
To further substantiate ETB-R-mediated apoptosis,
raSMC from 2- to 3-wk-old spotting lethal rats were cultured and tested
under the same experimental conditions. These animals lack a functional
ETB-R due to a 301 bp deletion at the border of
the first coding exon and the following intron, corresponding to the
NH2 terminus of the protein (18
, 19)
. Indeed, in raSMC from homozygous (sl/sl) animals, no
ET-1-mediated apoptosis occurred after exposure to cyclic strain, as
measured by the lack of increase in caspase-3 activity (Fig. 6C
). The overall basal level of apoptosis in these cells was
lower but still within the range of raSMC isolated from wild-type (+/+)
or heterozygous (sl/+) animals (Fig. 6A, B
). The pattern of
ET-1-induced ETB-R-mediated apoptosis was similar
in raSMC from sl/+ animals as compared to their +/+ littermates or
Wistar rats, but the overall induction of caspase-3 activity in these
cells was significantly lower, pointing to a gene dose effect.
Moreover, neither prepro-ET-1 nor ETA-R or
ETB-R expression appeared to be different in
raSMC from +/+, sl/+ or sl/sl animals from the same litter, as judged
by RT-PCR analysis (not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 21)
, an effect that may also be
related to an autocrine suppression by NO of endothelial ET-1 synthesis
(22)
. Enhanced cyclic stretch due to a chronic increase in
blood pressure, on the other hand, mainly affects the smooth muscle
cells and is thought to be involved in pressure-induced vascular
remodeling (23
24
25)
. As an enhanced synthesis of ET-1
seems to play a role in such adaptive responses of the vessel wall, we
examined whether rhythmic deformation affects expression of the
endothelin system differently in cultured endothelial and smooth muscle
cells.
Our findings with primary cultures of porcine endothelial cells confirm
earlier results of a stretch-induced increase in ET-1 release at both
the peptide and mRNA levels in bovine and human endothelial cells
(26
, 27)
, in human coronary arteries in situ
(13)
, and in rat carotid arteries after balloon
angioplasty (5)
. Moreover, we were recently able to
demonstrate a deformation-induced increase in ET-1-synthesis in the
endothelium but not in the smooth muscle of the isolated perfused
carotid artery and jugular vein of the rabbit (28
, 29)
.
Conversely, in cultured smooth muscle cells isolated from the rat
aorta, there was a marked decrease in ET-1 synthesis on exposure to
cyclic strain. If this finding can be extrapolated to the in
situ situation, it may represent a sensitization of the smooth
muscle cells to the enhanced release of ET-1 from the endothelium.
In addition to the decrease in ET-1 synthesis, there was a change in
the relative expression of the two endothelin receptors in the smooth
muscle cells on exposure to cyclic strain. Whereas
ETB-R mRNA levels were strongly up-regulated in
response to cyclic strain, ETA-R expression was
down-regulated but returned to control values within 6 h after
termination of stretch. Activation of both of these receptors
potentially triggers an identical biological response, as they are
coupled to the same trimeric G-proteins and, for example, elevate the
intracellular concentration of free calcium (30)
. However,
their biological effects seem to differ markedly, depending on the cell
type, tissue, and possibly the intracellular compartment in which they
are expressed (31)
. For example, predominant
ETB-R-mediated vasoconstriction is rarely seen in
arteries but seems to be an alternative mechanism by which ET-1 exerts
its constrictor effects in veins in vivo (32)
.
Similarly, in the present study we did not observe a pro-apoptotic
effect of exogenous ET-1 on nonstretched smooth muscle cells, i.e.,
where the ETA-R is presumably much more abundant
than the ETB-R. This pro-apoptotic effect became
apparent only after up-regulation of the ETB-R in
response to cyclic strain, a finding supported by earlier studies
describing an antiproliferative effect of stretch on cultured smooth
muscle cells from porcine aorta (33)
. Moreover,
preliminary evidence from this laboratory (29)
and two
previous reports (34
, 35)
suggest that in contrast to
arterial smooth muscle cells, cultured and native venous smooth muscle
cells do not undergo apoptosis on (rhythmic) deformation, but instead
increase their rate of proliferation in a BQ 788-sensitive manner,
pointing toward a phentotypic difference in the reaction of smooth
muscle cells to ETB-R signaling.
That the ET-1-induced apoptosis in stretched arterial smooth muscle
cells was indeed mediated through activation of the
ETB-R was evidenced by two findings. First, it
was completely abrogated in the presence of the
ETB-R antagonist, BQ 788, whereas the
ETA-R antagonist, BQ 123, had no or in some
experiments even an apoptosis-promoting effect. The latter finding
might be explained by the recovery of endogenous ET-1 synthesis in the
cultured raSMC 12 h after termination of the stretch protocol. In
addition, our data are in line with a recent report showing that
arterial smooth muscle cells undergo apoptosis in experimental
hypertension, an effect that is aggravated by
ETA-R blockade as well (36)
.
Therefore, activation of the ETA-R seems to
prevent rather than initiate apoptosis in arterial smooth muscle cells
of the rat.
Second, smooth muscle cells isolated from the aorta of homozygous
spotting lethal rats that lack a functional ETB-R
did not undergo apoptosis when exposed to both ET-1 and cyclic strain.
The spotting lethal rat is a crossbreed from Wistar-Kyoto rats with a
rat of unknown genetic background, carrying the aforementioned sl
mutation in the ETB-R gene (15
, 18
, 19)
. Whereas homozygous (sl/sl) animals die within the first 4
wk of life of a megacolon due to aganglionosis resembling human
Hirschsprung’s disease, heterozygous (sl/+) animals survive and seem
to be completely functional with regard to the endothelin system. In
fact, prepro-ET-1, ETA-R and even
ETB-R mRNA levels were comparable in raSMC
derived from +/+, sl/+, and sl/sl animals of the same litter; similar
findings have recently been obtained with astrocytes isolated from
wild-type and homzygous animals (15)
.
Moreover, the finding that raSMC cultured from homozygous as well as
heterozygous spotting lethal rats overall reveal a reduced
susceptibility to ET-1-induced apoptosis on exposure to cyclic strain
as compared to cells from wild-type animals or Wistar rats not only
highlights the role of a functional ETB-R in
stretch-induced apoptosis in arterial smooth muscle cells, but also
points to a gene dose effect in the spotting lethal rats. Recently,
such a gene dose effect has also been proposed at the level of the
endothelial ETB-R with the ET-3-induced relaxant
response of the basilar artery being lower in segments isolated from
heterozygous animals as compared to their wild-type littermates
(15)
.
Apoptosis has been reported to precede and even to be a prerequisite
for the development of hypertrophy in cardiomyocytes and thus
ventricular remodeling (37
, 38)
, reflecting not only a
change in the phenotype of preexisting cells, but possibly an exchange
of these terminally differentiated cells against cells with the
capacity to alter their phenotype. It is tempting to speculate,
therefore, that pressure-induced ETB-R-mediated
apoptosis of smooth muscle cells in the media marks the onset of
arterial remodeling in vivo.
In this context, the findings of the present study can be summarized as
follows (Fig. 7
). As long as blood pressure and thus cyclic strain are within the
physiological range, the ETA-R is the predominant
receptor expressed in arterial smooth muscle cells, mediating
ET-1-induced constriction and helping in this way to preserve the
existing geometry of the vessel wall. If blood pressure is elevated for
prolonged periods of time above the physiological level,
ETB-R expression is strongly enhanced, leading to
an increased rate of smooth muscle cell death. This phenotypic
alteration could mark the onset of vascular remodeling (i.e., medial
hypertrophy in conduit arteries and hyperplasia in resistance-sized
vessels) and reflect an adaptive response of the vessel wall to the
increased pressure load. Provided that the stimulus (i.e., the increase
in blood pressure) prevails, this will ultimately lead to fixation of
vascular resistance and thus to the manifestation of hypertension.
Although there can be no doubt that additional studies must corroborate
this hypothesis, our data provide a reasonable molecular mechanism,
from the physiological point of view, for these sensible adaptive
responses, which often tend to exaggerate and thus become
pathophysiologically relevant.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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REFERENCES |
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 J. Sanchez-Esteban, Y. Wang, L. A. Cicchiello, and L. P. Rubin Pre- and Postnatal Lung Development, Maturation, and Plasticity: Cyclic mechanical stretch inhibits cell proliferation and induces apoptosis in fetal rat lung fibroblasts Am J Physiol Lung Cell Mol Physiol, March 1, 2002; 282(3): L448 - L456. [Abstract] [Full Text] [PDF]
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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]
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H. D. Intengan and E. L. Schiffrin Vascular Remodeling in Hypertension: Roles of Apoptosis, Inflammation, and Fibrosis Hypertension, September 1, 2001; 38(3): 581 - 587. [Abstract] [Full Text]
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 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]
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 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]
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 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]
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