(Circulation. 1999;99:1147-1155.)
© 1999 American Heart Association, Inc.
Clinical Investigation and Reports |
Endothelin-1 and Its mRNA in the Wall Layers of Human Arteries Ex Vivo
From the Departments of Clinical and Experimental Medicine (G.P.R., S.C., E.P., G.A., F.D.R., P.P., A.C.P.), Cardiac Surgery (G.G., D.C.), and Biomedical Sciences and CNR Unit for Muscle Biology and Physiopathology (S.S.), University of Padova, Italy.
Correspondence to Gian Paolo Rossi, MD, FACC, Department of Clinical and Experimental Medicine, Clinica Medica 4, University Hospital, via Giustiniani, 2, 35126 Padova, Italy. E-mail gprossi{at}ux1.unipd.it
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Abstract |
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Background—The participation of endothelin-1 (ET-1) in the control of vascular tone in humans has been questioned, on the basis of the finding of subthreshold immunoreactive (ir) ET-1 plasma levels. However, because most ET-1 is secreted abluminally, it might attain a higher concentration in the tunica media than in plasma. Furthermore, evidence indicates that vascular smooth muscle cells (VSMCs) can synthesize ET-1 on stimulation in vitro. We therefore looked for irET-1 in the different layers of the wall of human arteries, including renal, gastric, and internal thoracic artery wall, obtained ex vivo from consenting patients with coronary artery disease and/or high blood pressure undergoing surgery, as well as from young organ donors.
Methods and Results—We performed immunohistochemistry with specific anti–ET-1 and anti-vWF antibodies followed by detection with an avidin-biotin complex ultrasensitive kit. The presence of preproET-1 and human endothelin-converting enzyme-1 (hECE-1) mRNA was also investigated by reverse transcription–polymerase chain reaction in homogenates of vessel wall, including preparations deprived of both endothelium and adventitia, and in isolated VSMCs. We detected irET-1 in the endothelium of all arteries and in the tunica media of internal thoracic artery from most patients with coronary artery disease. PreproET-1 and hECE-1 mRNA was also detected in VSMCs isolated from these vessels. irET-1 and irvWF staining in endothelium and tunica media was measured by use of microscope-coupled computer-assisted technology. Significant correlations between the amount of irET-1 in the tunica media and mean blood pressure (P<0.05), total serum cholesterol (P<0.05), and number of atherosclerotic sites (P<0.001) were found. Thus, in organ donors, irET-1 was detectable almost exclusively in endothelial cells, whereas in patients with coronary artery disease and/or arterial hypertension, sizable amounts of irET-1 were detectable in the tunica media of different types of arteries. In addition, VSMCs isolated from these vessels coexpressed the preproET-1 and hECE-1 genes.
Conclusions—Collectively, these findings are consistent with the contention that endothelial damage occurs in most patients with atherosclerosis and/or hypertension and that ET-1 is synthesized in VSMCs of these patients.
Key Words: arteries • endothelin • antibodies • RNA
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Introduction |
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Endothelins are a family of isopeptides with multiple biological actions that may be relevant for the regulation of vascular tone, blood pressure, and sodium and water homeostasis.1 2 3 Endothelin-1 (ET-1), originally isolated from the supernatant of porcine aortic endothelial cells, is the predominant isopeptide in the vascular endothelium.1 Because of its pleiotropic effects on the vasculature, it was hypothesized to intervene in the regulation of vascular tone under pathophysiological conditions and to play a role in the pathogenesis of hypertension, atherosclerosis, and related cardiovascular disease.4 5 6 7 However, this contention was challenged on the basis of the observation that although plasma levels of the peptide were increased in atherosclerotic patients and in hypertensives with cardiovascular disease,8 9 10 11 they were either only slightly increased or normal in most hypertensives.6 12 Furthermore, they are generally subthreshold, ie, lower than the 30 to 300 pmol/L that elicits a vasoconstrictor response in various smooth muscle (SM) preparations in vitro.5 7 In vitro studies with endothelial cell monolayers suggest that
80% of ET-1
would be secreted abluminally through the basolateral portion of
endothelial cells toward the tunica media, where it
could act in a paracrine fashion,13 14 but this
polarization was not found in the coronary vascular bed of
isolated rat hearts.15 In addition, it was reported that
vascular smooth muscle cells (VSMCs) could express the preproET-1 gene
in vitro under several experimental conditions,16 thereby
raising the possibility that this might also occur in vivo. We
therefore hypothesized that the peptide could be detected in the tunica
media of human vessels in vivo, where it was synthesized locally. Thus, our purposes were to investigate (1) the presence and distribution of immunoreactive (ir) ET-1 in the different layers of the wall of these vessels, (2) the expression of preproET-1 gene in the tunica media and in VSMCs of human arteries obtained ex vivo from young organ donors and from patients with coronary artery disease and/or high blood pressure, and (3) the clinical correlates of irET-1 in the arterial wall.
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Methods |
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Selection of Subjects
Consecutive otherwise healthy organ donors (n=5) and consenting patients undergoing coronary artery bypass graft surgery (n=19; age, 42 to 77 years), unilateral nephrectomy for kidney cancer (n=1), or surgery for lung (n=2) and gastric (n=1) cancer (age, 69 to 83 years) were investigated. The latter patients were selected so as to obtain different types of artery. Most arterial specimens were histologically normal, but 50% of those from coronary artery disease patients showed some intimal thickening.
In all patients, the presence and extension of atherosclerotic involvement was determined as described by Lerman et al.8 The study followed the local ethical guidelines for human investigations.
Preparation of Vascular Tissues
After excision, tissues were immediately frozen in isopentane,
cooled at -70°C over dry ice in the operating room, and stored at
-195°C in liquid nitrogen until they were used for analyses.
Frozen 7- to 10-µm sections were cut with a cryostat (Leitz 1720
Digital) at -20°C and used for the immunohistochemistry studies.
They were examined for localization of the mature ET-1 peptide and vWF
as a marker of the endothelium.
Immunohistochemistry for ET-1
To visualize the distribution of irET-1 peptide, the following
primary antibodies were used: rabbit anti–ET-1 antiserum (Peninsula
Laboratories Inc) that shows a 7% cross-reactivity for ET-2 and ET-3
and a 17% cross-reactivity for human big ET-1
(proendothelin-1),17 18 a monoclonal anti–ET-1 antibody
(IgG1)19 (ABR) directed against an epitope encompassing
amino acids 8 to 16 of ET-1. The latter antibody has <10%
cross-reactivity for ET-2 and <10% for ET-3, and no cross-reactivity
to nonrelated peptides. The reaction was detected with the Immunopure
ABC Ultra Sensitive Staining Kit (Pierce). Serial 10-µm slides of
mounted vessel sections were fixed in 10% formalin for 5 minutes.
Adjacent sections were incubated with an anti-vWF antibody (1:2000)
(Dako). Sections were washed in PBS (pH 7.4) and incubated with normal
goat serum (1/70) to block nonspecific binding for 20 minutes. Excess
serum was then removed and replaced with the primary antibodies
(1/500). Negative controls were carried out in all cases by treating an
adjacent section with preimmune rabbit or mouse IgG (1/500), either by
omitting the primary antibody or by preincubating it with a 10-fold
higher concentration of ET-1. Sections were incubated with primary
antibody at 37°C for 40 minutes. After a 10-minute wash, sections
were incubated at room temperature for 30 minutes with a secondary
biotin-conjugated goat anti-rabbit or anti-mouse IgG (1/200), with
normal human serum (1/200), and with goat serum (1/70). They were
rinsed again before incubation with avidin-biotin complex (30 minutes
at room temperature), specially purified avidin (1/60), and specially
prepared biotinylated horseradish peroxidase (1/60) constitute. The
reaction was developed for 10 minutes with 20 mg
3-amino-9-ethylcarbazole (Sigma Chemical Co), 2.5 mL
N,N-dimethyl formamide, 47.5 mL acetate buffer
(0.2 mol/L sodium acetate, 0.2N glacial acetic acid), and 25 µL 30%
H2O2 and was stopped with
tap water. The sections were mounted with polyvinyl alcohol
(Sigma).
Quantification of irET-1
For each artery section, 6 high-quality images were examined
with the microscope (magnification x20) and acquired on a personal
computer coupled to the microscope by use of specially developed
software (Studio Casti Imaging) under identical conditions of light in
black and white using a gray scale, white being set to a maximum
brightness value of 250 and black to 0. On each image, measurements on
endothelium, tunica media, and background, the latter
being defined as a tissue-free portion of the image in the vessel
lumen, were performed. The endothelium was identified
by comparison of each section with an adjacent one stained for vWF, the
tunica media being defined as the portion of wall between the internal
and external elastic laminae. The area of interest was outlined with a
cursor, and the average gray was measured. To exclude the confounding
effect of different backgrounds, the background value of each section
was subtracted from each reading. The average within-section
coefficients of variation of the measurement of irET-1 in the
endothelium and tunica media were 20% and 17%,
respectively.
Immunofluorescence Studies
The monoclonal SM-E7 anti-SM myosin antibody, which binds
selectively to SM-type myosin heavy chains (MHC) 1 and 2 (SM-1 and -2)
expressed in VSMCs,20 21 was used. Cryosections 7
µm thick from arteries and veins were fixed in 1.5%
p-formaldehyde, rinsed in PBS for 10 minutes at room
temperature, and then incubated at 37°C for 30 minutes with the
appropriate dilution of SM-E7 (IgG, 2.5 µg/mL) antibody. After being
rinsed with PBS, the sections were treated with a rabbit anti-mouse IgG
coupled with tetramethylrhodamine isothiocyanate (Dako) and mounted in
polyvinyl alcohol. The specimens were observed with an Olympus BX 50
fluorescence microscope. The reactivity to SM-E7 and
anti–
-actin (Sigma) antibodies was tested in cultured cells (see
below) at both an early and a late passage.
Immunofluorescence Studies of
Endothelin-1–Converting Enzyme in VSMCs
VP-16 polyclonal antibody was produced in rabbits immunized with
the 16-amino-acid peptide of the N-terminus endothelin-1–converting
enzyme (ECE-1a)–specific isoform coupled with keyhole limpet
hemocyanin. After affinity chromatography purification,
the antibody reacted exclusively with the 120-kDa electrophoretic band
contained in SDS extracts from cultured human umbilical vein
endothelial cells. The aforementioned monoclonal
anti–ET-1 was used. VSMCs were prepared from human aorta by use of a
standard procedure22 and used for
immunofluorescence experiments after the third
passage in vitro. Immunofluorescence assay was
performed on 1.5% p-formaldehyde–fixed and Triton
X-100–permeabilized cells. The primary antibodies
VP-16 and anti–ET-1, appropriately diluted, were mixed together and
incubated for 30 minutes in a humidified chamber. After being rinsed
with PBS, the cultures were incubated with a mixture of the secondary
antibodies, ie, anti-rabbit IgG coupled with FITC and anti-mouse IgG
coupled with rhodamine isothiocyanate (Dako) at the conditions
described above. Controls were nonimmune rabbit IgG followed by
anti-mouse IgG coupled with rhodamine isothiocyanate and nonimmune
mouse IgG followed by anti-rabbit IgG coupled with FITC.
SM Cell Cultures
Arteries collected in the operating room were immediately placed
in sterile Ringer's solution with penicillin and streptomycin at
4°C, cleaned of fat and connective tissue, and cut under a
stereoscopic microscope. The intima was scraped away and the adventitia
stripped away from the media in DMEM. The media was chopped fine and
rinsed in Hanks' balanced medium (Sigma), and the small chunks
obtained were placed in a small volume of medium containing 10% FCS,
200 mmol/L L-glutamine, penicillin 100 U/mL, and
streptomycin 100 µg/mL. Once outgrowth of cells was achieved, cells
were trypsinized and seeded into 25-cm2 Petri
dishes. Subconfluent cell cultures at the first passage were used after
being growth-arrested. Viability of cells was evaluated by dye
exclusion test with trypan blue. An aliquot of acetone-fixed cells was
used for immunofluorescence studies, and another
aliquot of 
250 000 cells was processed for RNA extraction with
RNAzol B (Duotech).
Gene Expression Studies
Samples of internal thoracic artery (ITA) (n=6) maintained on
ice were carefully deprived of both endothelium and
adventitia under a microscope. Reverse transcription–polymerase chain
reaction (RT-PCR) was performed from total RNA isolated by the
guanidinium isothiocyanate method by use of specific primers as
reported.3 23 Amplification was carried out for 38 cycles
at 94°C for 1 minute; 54°C and 60°C, as annealing temperature,
for preproET-1 and human ECE-1 (hECE-1), respectively, for 1 minute;
and extension at 72°C for 1 minute. To rule out the possibility of
amplifying genomic DNA, in some experiments the PCR was carried out
with no prior RT of the RNA. Detection of the PCR amplification
products was carried out by size-fractionation on 2% agarose gel
electrophoresis and ethidium bromide staining.
Statistical Analysis
Results are expressed as mean±SD or SEM. Comparisons were
carried out by 1-way ANOVA followed by Bonferroni's test or with
Kruskal-Wallis test for variables not normally distributed. To
investigate the relationship between demographic features, mean blood
pressure values, number of atherosclerotic sites, and irET-1, a
stepwise forward regression analysis (inclusion cutoff value of
0.05) was used.15 Pearson coefficient was also estimated
to assess the relationship of individual variables; a value of
P<0.05 was considered statistically significant. For
analyses, we used the SPSS for Windows statistical package
(version. 8.0, SPSS Inc).
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Results |
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Clinical Features of the Subjects
The demographic and clinical features of the subjects studied are summarized in Table 1
. In 16 cases
(67%), the indication for surgery was coronary artery disease,
and in 4 (17%) it was cancer. Arterial hypertension was
present in 58%, diabetes mellitus in 21%, and
hypercholesterolemia in 12% of the
subjects.
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Immunohistochemistry
Staining of the endothelium for irET-1 was seen in
all types of arteries, including hypogastric, mesenteric (Figure 1),
ITA, and femoral artery. However, in all
arteries from patients with coronary artery or other diseases,
irET-1 was detected not only in the endothelium
(Figures 2 and 3, arrows) but also in the tunica media.
The staining of the latter was homogeneous and involved
almost exclusively the muscle bundles, as confirmed by further
experiments carried out with combined immunocytochemistry for ET-1 and
immunofluorescence analysis for
VSMC-specific antibodies (see below). Similar results were obtained
with the monoclonal anti–ET-1 antibody (not shown). No staining was
observed in any negative controls carried out. The correlation matrix
and a regression analysis carried out with irET-1 in the tunica
media as dependent variable (Table 2)
revealed significant direct relationships between irET-1 in the media
and mean blood pressure levels, total serum cholesterol,
irET-1, and irvWF in the endothelium; an inverse
relationship between irET-1 in the media and number of sites involved
by atherosclerosis was also found.
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Immunofluorescence
As expected, all medial cells of cryosections were recognized by
the SM-E7. Almost all intimal cells also reacted to this antibody
(Figure 3D
), indicating that the thin intimal layer was composed
primarily of VSMCs. A few cells in the adventitia were stained with
this antibody, and in some cases, the anti–ET-1 antibody (not shown)
also recognized them. At the immunofluorescence
analysis, cultured cells were all positive to both the
anti–-actin and anti–SM-MHC antibodies, and with both antibodies,
the immunofluorescence pattern typical of VSMCs was
evident (not shown).
Double immunofluorescence experiments with
the monoclonal anti–ET-1 antibody and the anti–hECE-1 antiserum VP-16
showed the colocalization of irET-1 and hECE-1 in VSMCs isolated from
human aorta (Figure 4). No
immunofluorescence was seen in the control
experiments (not shown).
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Gene Expression
The RT-PCR allowed detection of the preproET-1 and hECE-1 mRNA in
all vessel specimens examined (Figure 5).
Amplified cDNA fragments of the expected size for both the preproET-1
(314 bp) and hECE-1 (567 bp) and for the control genes were easily
detected in samples of the normal arterial wall (Figure 5, top) and in preparations in which both
endothelium and adventitia were carefully removed
(Figure 5, top). No amplification was seen in the control PCR
containing either no cDNA (water) or total RNA without any prior RT,
thereby ruling out the possibility of false-positive results and of
amplification of genomic DNA, respectively. Hybridization with capture
probes specific for each cDNA investigated confirmed the specificity of
the amplification products for each mRNA (not shown). Of interest,
similar results for both the preproET-1 and the hECE-1 in VSMCs
isolated from ITA were found (Figure 5, bottom).
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Discussion |
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Our immunohistochemistry results show that ET-1 is found mainly on endothelium of different types of arteries from young, healthy normotensive individuals (Figure 1
). However, they also
show that irET-1 is detectable in the tunica media, and more precisely
on VSMCs, of human arteries obtained ex vivo from patients with
coronary artery disease and/or hypertension (Figures 2 and 3). This finding is in keeping with earlier reports on
atherosclerotic coronary arteries,24 25 26
transplant coronary arteries,27
atherosclerotic plaques,28 29 VSMCs isolated from
atherosclerotic coronary arteries,24 or
angioplasty-induced neointima formation.26
Several lines of evidence support the contention that irET-1 is
localized in VSMCs. First, the distribution of the
immunostaining within the tunica media of these
arteries is consistent with a predominant expression of irET-1
on SM bundles, ie, on VSMCs (Figures 2 and 3), and it is
well established that VSMCs are the only cell type present in the
normal tunica media.30 Second, the irET-1–stained cells
were conclusively identified as VSMCs by use of SM-E7, an antibody that
recognizes only VSMCs, regardless of their differentiative
state,20 21 and therefore stained all cells of the tunica
media (Figure 3). Rare VSMCs expressing ET-1 were also
recognized by the anti-myosin antibody in the adventitia close to the
external elastic lamina (Figure 3), but a few adventitial cells
expressing irET-1 (not shown) were not, thereby suggesting that
adventitial myofibroblasts can also express ET-1.
Thus, collectively, our ex vivo findings accord well not only with the
in vitro demonstration of a predominant abluminal secretion of
ET-113 14 but also with the contention that in patients
with coronary artery disease and/or hypertension, the peptide
is found in the tunica media, at variance with findings in otherwise
healthy young organ donors, in whom the peptide was detectable
primarily in the endothelium (Figure 1). Because
both ETA and ETB receptors
were found on VSMCs of the media,31 it might be that the
irET-1 detected in this layer is the
endothelium-derived peptide bound to its receptors.
However, the secretion of ET-1 is constitutive and the half-life of
secreted ET-1 in vivo is short (between 1.5 and 7 minutes), because it
is quickly inactivated in vascular cells,32
and the intracellular half-life of ET-1 mRNA is only 15
minutes.33 Thus, we propose that ET-1 is synthesized in
the tunica media, a hypothesis further suggested by 2 sets of evidence:
(1) the demonstration that ET-1 mRNA can be induced in vitro in human
VSMCs by growth factors and vasoactive
peptides16 18 24 27 28 29 34 35 36 and (2) the identification
of consensus sequences for a number of regulators, including shear
stress, the acute-phase reactant regulatory elements, transforming
growth factor-ß, insulin, thrombin, epinephrine,
interleukin-1, and angiotensin II, in the 5' flanking
region of the preproET-1 gene33 37 and of the hECE-1
gene.38 Therefore, to investigate this question further,
we sought the expression of the preproET-1 and hECE-1 genes in
homogenates of the vessel wall of ITA. We found that both
genes are expressed not only in homogenates of the wall of
these vessels23 but also, although less intensely, in
preparations of ITA that were carefully denuded in vitro of both the
endothelium and the adventitia (Figure 5, top).
Because endothelial cells on capillaries of the media
might account for these results, to prove that the preproET-1 and ECE-1
mRNA come from VSMCs, we isolated cells from ITA media and could show
that cultured VSMCs coexpress the preproET-1 and hECE-1 mRNA (Figure 5, bottom) at an early passage in culture. Thus, although we
cannot rule out the possibility that the preproET-1 gene is
activated once cells are placed in culture, our present
findings, as well as the demonstration that irET-1 is released by
cultured human VSMCs,24 suggest that ET-1 can be made by
VSMCs of the tunica media in vivo. This is further supported by the
demonstration of a colocalization of both immunoreactive ET-1 and
hECE-1 in VSMCs isolated and cultured from the human aorta (Figure 4).
Our arteries were obtained from a population with a
heterogeneous cardiovascular risk profile.
Most were from patients in their fifth decade and older who underwent
major surgical procedures for coronary artery disease or
cancer, and some were from younger organ donors. Thus, to gain insight
into the mechanisms linked to ET-1 biosynthesis in the
arterial wall layers, we carried out a regression
analysis (Table 2), which unveiled several interesting
significant relationships. First, a direct correlation between irvWF
and irET-1 in the endothelium was found, and an inverse
relationship between irvWF in the endothelium and the
number of sites involved by atherosclerosis was seen
(Figure 6). This finding indicates that
more extensive atherosclerotic involvement is associated with
progressive loss of endothelial expression of irvWF.
This inverse relationship, although statistically significant (Table 2), was less evident when irET-1 in the
endothelium was taken into consideration, suggesting
that the loss of irvWF, ie, of endothelial integrity,
is not closely paralleled by a decrease of ET-1 synthesis. We also
noticed a significant correlation between irET-1 in the
endothelium and in the media (Table 2). This
observation may suggest that the same factors are involved in
regulating the expression of the peptide in both wall layers. However,
we found that irET-1 in the media was directly related
(P<0.05) to the level of mean blood pressure and total
serum cholesterol, 2 well-known
cardiovascular risk factors, whereas irET-1 in the
endothelium was not. We therefore suggest that although
endothelial irET-1 is subjected to short-term
regulation and/or to loss of endothelial lining
integrity, the ET-1 in the media constitutes a more stable pool. A
contribution of aging cannot be conclusively ruled out at this stage,
but our analysis did not provide support to the contention of a
relationship of irET-1 with age either in the
endothelium or in the tunica media. The fact that
irET-1 in the media was inversely related to the extension of
atherosclerosis, ie, to the number of atherosclerotic
sites, is intriguing in view of the previous finding of a direct
relationship between plasma levels of irET-1 and sites of
atherosclerosis.8 However, it may
indicate either a more important role of ET-1 in the initiation rather
than in advanced stages of atherosclerosis and/or an
increased turnover of ET-1 at the vascular wall level leading to
increased plasma levels of the peptide.
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Our findings, along with the detection of preproET-1 and ECE-1 mRNA in the tunica media and in isolated VSMCs, suggest that ET-1 synthesis can be activated in vivo in the VSMCs of ITA and other types of arteries from patients with and without coronary artery disease and/or hypertension. Nonetheless, they speak against an exclusive activation of ET-1 biosynthesis in endothelial cells under most pathophysiological conditions, a conclusion that accords well with the results of other24 25 28 but not all investigators.26
It has recently been shown that blockade of both endothelin receptors with the nonselective agent TAK-044 and inhibition of hECE-1 with an intravenous infusion of phosphoramidon cause peripheral vasodilatation in humans.39 40 It was therefore proposed that in vivo generation of ET-1 plays a pivotal role in the maintenance of vascular tone. By showing an ET-1 biosynthesis in the tunica media, our findings provide a rational explanation for these hemodynamic responses.
In conclusion, we found that the preproET-1 and hECE-1 genes are expressed in the tunica media and isolated VSMCs and that irET-1 is detectable not only in the endothelium but also in muscle bundles of the tunica media from histologically normal human arteries obtained ex vivo from patients with different diseases. Thus, these findings are consistent with an important role of ET-1 in the autocrine-paracrine regulation of vascular tone, as recently confirmed by observations in normotensive and hypertensive humans.39 40 41 42 43
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Acknowledgments |
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We gratefully acknowledge the valuable collaboration of all our colleagues of the Departments of Cardiac Surgery (directed by Prof Dino Casarotto) and of Drs Umberto Tedeschi, Umberto Cillo, Giacomo Zanus, and Enrico Gringeri of the P.G. Cevese Institute of Clinical Surgery 1 (directed by Prof Davide D'Amico) of the University of Padua.
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Footnotes |
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Guest Editor for this article was Paul J. Cannon, MD, College of Physicians and Surgeons, New York, NY.
Received May 10, 1998; revision received November 4, 1998; accepted November 23, 1998.
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