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Endothelin an endothelial-dependent vasoconstrictor in scleroderma. Enhanced production and profibrotic action

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Enhanced Production and Profibrotic Action
The vascular endothelium is an important functional unit in the regulation of the vascular and perivascular environment. Various chemical and physical stimuli mediate an endothelial-dependent vasoconstriction
through the release of endothelial soluble factors, such
as the recently recognized endothelium-derived vasoconstrictor peptide called endothelin. The presence of circulating endothelin and the effect of cold exposure on
plasma endothelin levels were investigated in patients
with scleroderma and in healthy control subjects. Radioimmunoassay demonstrated a mean f SD plasma
level of 10.7 f 7.3 pg/ml in the patients (n = 19) and 3.7
f 2 in the control subjects (n = 16) (P< 0.005). These
levels were also assessed in 5 control subjects and 5
scleroderma patients before and after 30 minutes of total
body cooling (to 15OC). The endothelin level did not
change significantly in either group; however, 2 scleroderma patients showed a significant increase after cooling. The effects of endothelin on fibroblast proliferation
and collagen synthesis were evaluated in order to assess
the impact of released endothelin on the interstitium. A
significant mitogenic effect and a collagen synthesisenhancing effect, which were dose-dependent, were
seen. The strong, characteristically prolonged, vasoconstrictor activity coupled with the profibrotic effect demonstrated here make it likely that disturbances in the
From the Medical College of Ohio, Toledo.
Supported by a grant from the Scleroderma Research
M. Bashar Kahaleh, MD.
Address reprint requests to M. Bashar Kahaleh, MD,
Medical College of Ohio, 3000 Arlington Avenue, PO Box 10008,
Toledo, OH 43699-10008.
Submitted for publication September 10, 1990; accepted in
revised form February 7, 1991.
Arthritis and Rheumatism, Vol. 34, No. 8 (August 1991)
control of endothelin production can contribute to the
pathogenesis of scleroderma.
Recent research on the vascular endothelium
has established the endothelial cell as an active cell
type which performs a variety of critical functions that
affect the vascular and interstitial biology and pathology. Vasospasm, reduced microvascular blood flow,
and increased vascular resistance are some of the
recognized abnormalities of the physiology of the
vascular system in scleroderma. The mechanism of
abnormal vascular function is not known. Endothelin,
a recently described endothelial-dependent vasoconstrictor peptide, is thought to play an important role in
the regulation of blood flow in the microvascular beds
(1). It has recently been shown that at low concentrations, endothelin produces a predominant vasodilatation, whereas at high concentrations, a vasoconstrictor response is seen (2). Endothelin interacts with a
specific receptor on smooth muscle cells and induces
an influx of Ca2+, which leads to vasoconstriction (3).
In view of this profile of biologic action, it
seemed plausible that endothelin could be involved in
the development of the functional vascular disorder in
scleroderma. It is also believed that released endothelial products might be involved in the initiation of
tissue fibrosis. Indeed, recent studies have demonstrated that, early in the course of scleroderma, activated fibroblasts expressing high levels of types I and
111 collagen messenger RNA (mRNA) are present
adjacent to blood vessels; this suggests the possible
occurrence of a vascular-related event that mediates
fibroblast activation and tissue fibrosis (4). In the
present study, plasma levels of circulating endothelin
were measured in patients with scleroderma, and the
effects of endothelin on normal dermal fibroblast
growth and collagen synthesis were examined.
Study population. Nineteen patients with diffuse
scleroderma and 16 healthy age- and sex-matched control
subjects were studied. All patients had normal renal function
and normal blood pressure at the time of study. Antiinflammatory, vasoactive, and diuretic agents, as well as other
agents which might affect the sympathetic nervous system,
were withheld for at least 2 weeks prior to study. All patients
had extensive skin involvement, with a mean skin score of 28
(range 2 2 4 8 , maximum possible score 66) (5). The mean
disease duration was 3.1 years, with a range of 0.64.0 years.
Blood samples were drawn into 1/10 volumes of 0.13
moles/liter of trisodium citrate in siliconized vacutainer
tubes (Becton-Dickinson, Mountain View, CA). Plateletpoor plasma was prepared as soon as possible by centrifuging blood samples for 10 minutes at 2,OOOg. The plasma was
stored in aliquots at -20°C until analyzed.
Cold exposure. Five control subjects and 5 scleroderma patients were studied. On the test morning, subjects
were placed in a room maintained at 23°C and seated in a
semirecumbent (45") position. An 18-gauge plastic catheter
was inserted into an antecubital vein and connected by
standard tubing and 3-way stopcock to a 5% dextrose
solution. Subjects were allowed to acclimate for 30 minutes,
and blood samples were drawn. Subjects were then transferred to an environmentally controlled room, where temperature, humidity, and sound are kept constant; the temperature is maintained at 15°C. After 30 minutes of total
body cooling, blood samples were drawn.
Endothelin assay. Plasma samples (1 ml each) were
acidified by adding 1 ml of 0.1% trifluoroacetic acid (TFA).
This solution was applied to a Sep-Pak C18 column (Peninsula Laboratories, Belmont, CA) that had been activated
with 60% acetonitrile in 0.1% TFA (4 ml) and washed with
0.1% TFA (20 ml). The column was then washed with 20 ml
of 0.1% TFA and eluted with 3 ml of 60% acetonitrile in 0.1%
TFA. The eluate was evaporated in a speed vacuum, and
was stored at -70°C until assayed.
A radioimmunoassay kit (Peninsula) was utilized to
measure endothelin levels. The assay is based on competitive binding of antiendothelin antibody by '2SI-labeled endothelin (porcine) and a standard concentration of unlabeled
endothelin (porcine). The minimum detectable level is 1
pg/ml. The antibody used in this assay cross-reacts fully with
endothelin 1, 2, and 3, but not with big porcine or human
endothelin or non-endothelin peptides (6,7). The samples
were stable, without detectable degradation, after 3 months
of storage at -70°C. There was no significant correlation
between the period of storage and the level of endothelin in
the stored plasma. The interassay and intraassay coefficients
of variation (n = 5) were 10% and 13%, respectively.
Fibroblast growth. Fibroblasts were obtained from
4-mm punch biopsies of skin from the dorsum of the forearm
of normal volunteer donors. At the fifth subpassage, cells
were cultured in microtiter wells (Falcon, Oxnard, CA) at
10,000 cells/well in low serum concentration (0.5%) for 48
hours. Endothelin (synthetic endothelin 1; Peninsula), in
serial dilution, was added, and cultures were continued for
72 hours. 3H-thymidine was added, at 0.5 pCilwell, during
the last 18 hours, and the cells were harvested and counted
in a scintillation counter.
Collagen and protein synthesis assays. Normal dermal
fibroblasts were exposed to 2.3 pCi of 3H-proline (20 Ci/
mmole; New England Nuclear, Boston, MA) in fresh medium (without serum) for 3 hours at 37°C. In a modification
of the collagen assay method described by Peterkofsky and
Diegelmann (8),cells were removed with a rubber policeman, and the cells and the supernatants were placed on ice
and precipitated overnight with cold 10% trichloracetic acid
(TCA) in the presence of 0.02% unlabeled proline and 4
mg/ml of bovine serum albumin (Cohn fraction V; Sigma, St.
Louis, MO). After 5 washings in 5% TCA and 0.01% proline
at 4"C, the precipitate was dissolved in 0.2N NaOH, and
exposed for 1 hour at 37°C to Tris-CaC1, buffer (0.05M Tris
HC1, pH 7.6, O.OO5M CaCI,, and 0.02% NaN,) alone, or to
Tris-CaCI, buffer containing either purified bacterial collagenase (repurified by gel filtration chromatography until
negative for protease, using chick protein labeled with
3H-tryptophan [9]; Worthington, Freehold, NJ), or pronase
(4 mg/ml; Calbiochem, La Jolla, CA). All samples were
incubated in the presence of N-ethylmaleimide (2.5 mM).
The test samples were placed on ice and precipitated
a second time for 1 hour with 10% TCA and 0.5% tannic
acid. The precipitate was discarded, and a 0.5-ml aliquot of
the supernatant was added to 0.5 ml of water and 10 ml of
scintillation fluid (8.5 gm of Omnifluor [New England Nuclear]
dissolved in I liter of toluene and mixed with 500 ml of Triton
X-100). Samples were counted in a Beckman (Fullerton, CA)
scintillation counter for 20 minutes. Counts obtained from
parallel test plates were used to calculate the amount of
collagen and protein synthesized per cell, as described
elsewhere (8,9).
Plasma levels of endothelin. Endothelin was
detected in plasma samples from all study subjects,
thus supporting the concept that endothelin is a circulating hormone. There was considerable overlap in
plasma endothelin levels between scleroderma patients and control subjects (Figure l). The mean (+SD)
level in the scleroderma group, however, was significantly higher than that in the control group (10.7 k 7.3
pg/ml versus 3.7 k 2 pg/ml; P < 0.005), which indicates an overproduction and/or a reduced clearance of
endothelin in some of the scleroderma patients.
Effect of exposure to cold. The mean (2SD)
plasma level prior to cold exposure was significantly
higher in the scleroderma group (8.3 k 2.3 pg/ml) than
in the control group (2.9 '-c 1 pg/ml) (Table 1). After 30
minutes of total body cooling at 15"C, all of the
patients, but none of the control subjects, had visible
Raynaud's phenomenon. The mean endothelin plasma
mean 2 SO
10.7 2 7.3'
mean 2 SD
3.7 f 2
Figure 1. Plasma endothelin levels in scleroderma patients and ageand sex-matched healthy control subjects. Values are the mean of
triplicate determinations for each plasma sample.
level was not significantly different from the baseline
level in either group. However, in scleroderma patients l and 4, there was a significant increase in the
plasma endothelin level after cooling; in patient 2 , the
Table 1. Plasma levels of endothelin in 5 scleroderma patients and
5 normal control subjects, before and after total body cooling*
Scleroderma patients
Subject 1
Subject 2
Subject 3
Subject 4
Subject 5
Mean %
Control subiects
8.3 ? 2.3t
9.2 f 5.8t
* Subjects were exposed to a 15°C room for 30 minutes, as described
in Patients and Methods. Values are in pgiml.
t P < 0.005 versus controls.
endothelin level decreased after cooling. After exposure to cold, the endothelin levels in the scleroderma
group were still significantly higher than those in the
control group ( P < 0.005).
Effect on fibroblast growth. Low levels of 3Hthymidine incorporation were noted when cells were
maintained in low concentrations (0.5%) of serum. The
addition of endothelin resulted in a dose-dependent
enhancement of thymidine incorporation that plateaued at high concentrations of endothelin. Figure 2
shows a typical growth-response curve of the fibroblast lines. Five different cell lines were tested, and all
showed similar responses, with a mean increase in
3H-thymidine incorporation of 310 + 25% above control values at an endothelin concentration of 100 pg/ml.
The difference between the values at baseline and Sfter
endothelin addition was significant at P < 0.001.
Effect on protein and collagen synthesis. Endothelin induced an increase in the synthesis of both
protein and collagen by fibroblasts in a dosedependent manner (Figure 3). However, the concentrations of endothelin needed to demonstrate this
effect were higher than those needed to produce the
mitogenic effect. The effect on protein and collagen
synthesis was seen in all cell lines tested. The mean
(+SD) percentages of increase in collagen and protein
synthesis at 10 ng/ml of endothelin were 60 -+ 5% and
71 t 18%, respectively, above baseline values in the 5
cell lines tested. The differences in protein and colla-
98 1
r =
Figure 3. Protein and collagen synthesis by normal dermal fibroblasts after 72 hours of incubation with various concentrations of
endothelin. Values are the mean 2 SD of quadruplicate experiments, and are expressed as pronase or collagenase releasable
counts per cell x 10 - 4 .
gen synthesis at baseline and after addition of endothelin were statistically significant at P < 0.005.
Research on the vascular endothelium in recent
years has established that endothelial cells are active
cells that not only control their own environment but
can also influence the surrounding tissues. This effect
is achieved through the capacity of the endothelial
cells to respond to various stimuli by undergoing
specific alterations in their functions, characterized by
an increase or decrease in normal constitutive functions, or by inducing new functions and molecules.
One of the endothelial-derived mediators is the potent
vasoconstrictor peptide, endothelin, which was recently purified and sequenced by Yanagisawa and
coworkers (10). Endothelin is shown to induce, in a
dose-dependent manner, the contraction of vascular
smooth muscle cells, which develops slowly and
reaches maximum contraction within 15-20 minutes of
initiation. The calcium channel blockers markedly
attenuate this response ( 1 1 ) . Endothelin also has a
potent constrictor action on the microvasculature,
leading to a decrease in basal blood flow and a reversal
of the increase in blood flow induced by vasodilators
(1). Endothelin may be involved in coronary vasospasm, as indicated by the delayed filling of the
coronary circulation following infusion (12). Renal and
pulmonary vasoconstriction is seen following systemic
infusion of endothelin, indicating a significant role for
endothelin in the regulation of peripheral vasomotor
tone (2).
In the present study, endothelin was detected in
plasma from normal subjects, which suggests the
presence of a circulating pool of endothelin. This
finding supports the concept that endothelin may be a
circulating hormone (13). The level of immunoreactive
endothelin in human plasma has been reported to be
1.59 pg/ml, as measured by a sensitive sandwich-type
enzyme-linked immunosorbent assay (14); this level is
similar to that found in the present study. Increased
plasma levels of endothelin have been reported in
patients with essential hypertension ( 1 3 , ischemic
heart disorders ( 1 6), and cyclosporine-induced nephropathy (17).
The elevation in plasma endothelin levels in
scleroderma patients reported here suggests an increased production of endothelin by the vascular
endothelium in scleroderma. In view of the known
prolonged vasoconstrictor effect, the possibility that
endothelin mediates the vasospastic episodes of
Raynaud’s phenomenon was investigated. The coldexposure experiment, as performed in this study, did
not influence the plasma level of endothelin, despite
the induction of Raynaud’s phenomenon in all patients. A more detailed study utilizing local cooling of
the hands and frequent sampling of the venous blood
of the hands will be necessary for any determination
that endothelin is involved in the pathogenesis of
Raynaud’s phenomenon. However, it can be argued
that the high rate of production of endothelin in
scleroderma may predispose to vasospasm by counteracting the effects of the natural vasodilatory mechanisms (1). The production by the endothelium of
endothelin and endothelium-derived relaxing factor, 2
substances with opposite vascular effects, suggests a
complex role for the endothelium in the regulation of
vascular tone, and implies the need for a systematic
evaluation of the mechanisms of vasoconstriction and
vasodilation in order to understand the pathophysiology of Raynaud’s phenomenon.
The finding in this study of a potent mitogenic
effect of endothelin on human diploid fibroblasts supports the data in published studies describing the
effects of endothelin on a variety of cell types. In
quiescent cultures of rat aortic smooth muscle cells,
endothelin was shown to rapidly elevate mRNA levels
of c-fos and c-myc in association with increased 3Hthymidine incorporation into DNA (18). This effect is
dependent in part on protein kinase C. Endothelin may
act synergistically with epidermal growth factor and
transforming growth factor a (19). Platelet-derived
growth factor (PDGF) at submitogenic concentrations
can enhance endothelin’s mitogenic effect on smooth
muscle cells (20). High-affinity receptors for endothelin have been identified in the murine cell line, 3T3,
and in pericytes (21,22). Receptor binding leads to
cellular proliferation, which can be inhibited by the
calcium channel blockers (19).
In scleroderma, diffuse organ fibrosis is prominent and is closely associated with alterations of the
small arteries and microvessels (23). The relationship
between connective tissue deposition and vascular
abnormalities is poorly understood. A growing body of
evidence suggests that the increased synthesis of connective tissue in scleroderma is the result of augmented collagen synthesis by dermal fibroblasts (24).
However, the mechanism by which fibroblast collagen
synthesis becomes augmented is unknown. Histologic
studies of early skin lesions in scleroderma patients
have revealed increased numbers of fibroblasts (25).
Fibroblast cultures established from explants of dermis from scleroderma patients have shown increased
collagen accumulation (26). Cultures of lower dermal
fibroblasts accumulate more collagen than do those
from the upper dermis (27); this suggests a possible
association between fibroblast synthesis and vessel
Recent studies have demonstrated that, early in
the course of scleroderma, activated fibroblasts expressing high levels of types I and I11 collagen mRNA
are located adjacent to blood vessels that are surrounded with mononuclear cells, suggesting a close,
and possibly causal, relationship (4). A soluble mediator resulting from interactions of endothelial and
mononuclear cells may directly activate fibroblasts to
migrate, proliferate, and produce excess amounts of
matrix. Involvement of PDGF in this process is suggested by the finding of intense expression of PDGF by
the endothelial lining of small capillaries in scleroderma patients (28). Observations in this study suggest
that endothelin is a possible contributor to this process, acting alone or in synergy with other cytokines
and growth factors to activate fibroblasts. Transforming growth factor p, a potent inducer of fibrosis,
strongly stimulates the expression of endothelin
mRNA by the vascular endothelium (12,29). Endothelin may eventually prove to be the link between
vascular pathology, Raynaud’s phenomenon, and the
pathologic deposition of connective tissue matrix in
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production, endothelial, vasoconstriction, profibrotic, action, enhance, dependence, scleroderma
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