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Vol. 55, Issue 5, 902-909, May 1999
Vascular Inflammation,
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Summary |
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 Top
 Summary
 Introduction
Experimental Procedures
Results
Discussion
References
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Endothelin-1 (ET-1) is the predominant endothelin isopeptide generated
by the vascular wall and therefore appears to be the most important
peptide involved in regulation of cardiovascular events. Many
pathologic conditions are associated with elevations of ET-1 in the
blood vessel wall. Because these conditions are often cytokine driven,
we examined the effects of a mixture of cytokines on ET-1 production in
human vascular smooth muscle cells (VSMCs) derived from internal
mammary artery and saphenous vein (SV). Incubation of IMA and SV VSMCs
with tumor necrosis factor-
(10 ng/ml) and interferon-
(1000 U/ml) in combination for up to 48 h markedly elevated the
expression of mRNA for prepro-ET-1 and the release of ET-1 into
the culture medium. This cytokine-stimulated release of ET-1 was
inhibited by a series of dual endothelin-converting enzyme
(ECE)/neutral endopeptidase inhibitors, phosphoramidon, CGS 26303, and
CGS 26393, with an accompanying increase in big ET-1 release but with
no effect on expression of mRNA for prepro-ET-1. These same compounds
were 10 times more potent at inhibiting the conversion of exogenously
applied big ET-1 to ET-1. ECE-1b/c mRNA is present in SV VSMCs, however
no ECE-1a is present in these cells. Thus VSMCs most probably
contain, like endothelial cells, an intracellular ECE responsible for
the endogenous synthesis of ET-1. Under the influence of
pro-inflammatory mediators the vascular smooth muscle can therefore
become an important site of ET-1 production, as has already been
established for the dilator mediators nitric oxide, prostaglandin
I2, and prostaglandin E2.
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Introduction |
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Top
Summary
Introduction
Experimental Procedures
Results
Discussion
References
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In
recent years it has become clear that following exposure to cytokines
and/or endotoxin vascular smooth muscle releases vasoactive mediators
normally produced within the vascular endothelium. Most notable,
perhaps, among these is nitric oxide (NO), which is produced within the
vascular smooth muscle following expression of inducible NO synthase
(iNOS) (Wong and Billiar, 1995
). Similar stimuli also induce the smooth
muscle to express cyclo-oxygenase-2, thereby increasing its capacity to
produce the vasodilator prostaglandins E2 and
I2 (Bishop-Bailey et al., 1997a,b). Apart from
vasodilator mediators, the normal vascular endothelium also produces
the potent vasoconstrictor and pro-mitogenic peptide endothelin-1
(ET-1) (Yangisawa et al., 1988; Inoue et al., 1989; Haynes and Webb, 1993; Warner et al., 1996; Parris and Webb, 1997). As for NO and the
prostaglandins, an increased production of ET-1 within the blood vessel
has been implicated in the events underlying a number of vascular
pathologies (Haynes and Webb, 1993; Warner et al., 1996; Parris and
Webb, 1997). For example, both specific ET receptor antagonists and
ET-1 binding antibodies produce beneficial effects in experimental
models of ischemia/reperfusion injury, restenosis, and hypertension as
well as in similar human disease states (Haynes and Webb, 1993; Warner
et al., 1996; Parris and Webb, 1997). Interestingly, in a number of
these vascular pathologies, most obviously in restenosis following
balloon angioplasty, there is loss or dysfunction of the endothelium,
yet ET-1 still appears to be involved as a causative agent (Haynes and
Webb, 1993; Warner et al., 1996; Parris and Webb, 1997). Drawing on our
experience of the NO and prostaglandin systems, this suggests that it
could be the vascular smooth muscle that produces ET-1 when the
endothelium is dysfunctional (Wang et al., 1996). Knowing that it is
pro-inflammatory cytokines that induce the expression of NO and
prostaglandins in smooth muscle, we might expect that it is these same
mediators that induce the expression of ET-1. Indeed, many of the
disease states in which ET-1 appears involved are associated with
elevations in the plasma concentrations of pro-inflammatory cytokines
such as tumor necrosis factor- (TNF-) and interferon-
(IFN-) (Warner and Klemm, 1996). In this study, we used vascular
smooth muscle cells (VSMCs) derived from human internal mammary artery
(IMA) and saphenous vein (SV) to examine the effect of cytokines upon
the production of ET-1. Because it appears that in endothelial cells
ET-1 is formed from its precursor big ET-1 by the action of an
intracellular endothelin-converting enzyme (ECE), we also used
inhibitors of ECE to characterize the synthetic pathway present within
cytokine-treated VSMCs (Corder et al., 1993; Schmidt et al., 1994;
Shimada et al., 1994; Xu et al., 1994). Some of this data was presented
to the Fifth International Meeting on Endothelin (Woods et al., 1998).
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Experimental Procedures |
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Top
Summary
Introduction
Experimental Procedures
Results
Discussion
References
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IMA and SV were obtained from patients undergoing coronary artery bypass surgery.
Cell Culture.
VSMCs were obtained for culture using the
free-floating explant method (Ross, 1971). Vessels were first cleaned
and then opened longitudinally and scraped to remove endothelial cells.
The smooth muscle was stripped from the adventitia and cut into
approximately 2-mm2 pieces before being placed
into tissue culture flasks with Dulbecco's modified Eagles medium
containing 2 mM glutamine, nonessential amino acids, sodium pyruvate,
and 15% fetal calf serum. Cells were then incubated at 37°C in 5%
CO2/95% air. After 4 to 6 weeks in culture,
sufficient cells had explanted to allow passage of cells for
characterization and use in experiments. Smooth muscle cells (passages
2-9) were identified by smooth muscle -actin staining. Endothelial
cell contamination was excluded by the absence of staining for von
Willebrand factor.
Experimental Procedures.
Throughout all experiments cells
were grown in Dulbecco's modified Eagles medium containing 2 mM
glutamine, penicillin (100 U/ml), streptomycin (100 µg/ml), and 10%
fetal calf serum. In the first series of experiments IMA and SV VSMCs
were grown on 96-well plates to confluence (approximately 2500 cells/well) and concentration-response curves to both TNF-
(0.01-100 ng/ml) and IFN- (0.1-1000 U/ml) were constructed in IMA
and SV. The TNF- concentration-response curve was carried out in the
presence of a fixed concentration of IFN- (1000 U/ml) and the
IFN- concentration-response curve was carried out in the presence of
a fixed concentration of TNF- (10 ng/ml). From these first
experiments, cytokine concentrations that produced maximal stimulation
of ET-1 release were identified.
(10 ng/ml) and IFN- (1000 U/ml) for the final 1, 4, 8, 12, 24, 36, or
48 h of a 48-h incubation period. The medium was retained for
assay of ET-1 content and the cells removed for analysis of expression
of mRNA for prepro-ET-1, ET-2, and ET-3.
In the third series of experiments, VSMCs grown on 96-well plates were
incubated with a combination of TNF- (10 ng/ml) and IFN- (1000 U/ml) for 48 h in the presence of a series of ECE and/or neutral
endopeptidase 24.11 (NEP) inhibitors; phosphoramidon, CGS 26303, CGS
26393 (a prodrug for CGS 26303), or CGS 24592 (1 nM to 1 mM) (De
Lombaert et al.,1994; Trapani et al., 1995).
To examine the effects of ECE inhibitors on the conversion of exogenous
big ET-1, VSMCs from both IMA and SV were treated with cytokines as
above for 24 h, the medium replaced, and cells incubated with big
ET-1 (1 µM) for 2 h in the presence of phosphoramidon, CGS
26303, CGS 26393, or CGS 24592 (1 nM to 1 mM). The effects of ECE
inhibitors on expression of prepro-ET-1 mRNA was determined by growing
cells on 75-cm2 flasks, which were then treated
with TNF- (10 ng/ml) and IFN- (1000 U/ml) in the presence or
absence of either phosphoramidon (300 µM), CGS 26393 (300 µM), or
CGS 26303 (1 mM) for 48 h. The cells were then removed for
analysis of expression of mRNA for prepro-ET-1.
All experiments were carried out in the presence of a cocktail of
peptidase inhibitors, captopril (1 µM), bestatin (1 µM), thiorphan
(1 µM), and bacitracin (3 µg/ml) to prevent degradation of ET-1 by
endogenous peptidases.
Measurement of Immunoreactive ET-1 and Big ET-1.
Medium was
removed and stored at 
70°C until further use. ET-1 and big ET-1
levels were measured using specific sandwich immunoassay kits following
manufacturers' instructions. Optical density at each appropriate
wavelength was measured using a Molecular Devices microplate reader
(Molecular Devices, Menlo Park, CA). Values are reported as mean ± S.E.M. and are representative of cells from four to five patients
each assayed in triplicate.
Cellular Respiration/Viability. Cell respiration, an indicator of cell viability, was assessed by the ability of cells to reduce 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to formazan. At the end of each experiment, cells in 96-well plates were incubated with MTT (1 mg/ml) at 37°C for 1 h. Following removal of MTT by aspiration, cells were solubilized in dimethyl sulfoxide (200 µl). The extent of reduction of MTT to formazan was quantified by measurement of optical density at 550 nm.
Expression of mRNA.
VSMCs from both SV and IMA were grown to
confluence in 75-cm2 flasks. Cells were lysed in
denaturing solution and total RNA was isolated by a guanidinium
thiocyanate/isopropanol method with minor modifications (Chomczynski
and Sacchi, 1987). Before cDNA synthesis, RNA was treated with
commercially available deoxyribonuclease I to eliminate any
contaminating DNA. One microgram of total RNA was then converted to
cDNA using Ready-To-Go T-primed First Strand Kit. The cDNA obtained
from this reaction was used in reverse transcription-polymerase chain
reaction (RT-PCR). RT-PCR was performed with an OmniGene Hybaid
thermocycler (Hybaid, London, UK. Initial denaturation was
performed at 94°C for 2 min, followed by annealing for 45 s at
the appropriate temperature and extension at 72°C for 1 min for 35 to
40 cycles, with the final extension period of 7 min at 72°C. The
annealing temperatures were set as follows: prepro-ET-1, 53°C;
prepro-ET-2, 58°C; prepro-ET-3, 53°C; glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 60°C. Gene specific primers were selected according to the published sequences in Genbank (see Table
1). GAPDH, a constitutively expressed
gene, was used as a positive control to correct for intersample
variation.
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Materials.
DMEM plus sodium pyruvate, fetal calf serum,
penicillin, streptomycin, glutamine, nonessential amino acids, sodium
pyruvate, and deoxyribonuclease I were supplied by Gibco BRL (Paisley,
UK) and bacterial endotoxin were obtained from Sigma Chemical Co. (Poole, UK). Human recombinant TNF-, IFN-, interleukin-1
,
interleukin-6, and human ET-1 immunoassay kit were purchased from R&D
Systems (Abingdon, UK). Human big ET-1 peptide and phosphoramidon were obtained from The Peptide Institute (Osaka, Japan). Big ET-1
immunoassay kits were purchased from Assay Designs, Inc. (Ann Arbor,
MI). CGS 26303 ((S)-2-biphenyl-4yl-1-(1H-tetrazol-5-yl)-ethylamino-methyl phosphonic acid), CGS 26393 (prodrug for CGS 26303), and CGS 24592, ((S)-N-[2-(phosphonomethylamino-3-(4-biphenyl)-propionyl]-3-aminopropionic acid) were a generous gift from Dr. Arco Jeng (Novartis, Summit, NJ). Total RNA extraction kit was purchased from Clontech
Laboratories, Inc. (Palo Alto, CA). Access RT-PCR kit was purchased
from Promega (Southhampton, UK) and the RNAzol B was purchased from
Biogenesis (Poole, UK). The first-strand cDNA synthesis kit
(Ready-To-Go T-Primed First Strand Kit) was obtained from Pharmacia
Biotech (Hertfordshire, UK). Primer sequences were synthesized by
Severn Biotech Ltd. (Worcestershire, UK).
Statistical Analysis. Data is expressed as mean ± S.E.M. Statistical comparisons were made by ANOVA or unpaired t test and values were considered to be significant at p < .05.
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Results |
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Top
Summary
Introduction
Experimental Procedures
Results
Discussion
References
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Concentration-Dependent Effects of TNF- and IFN- on
Production of ET-1 by Human IMA and SV VSMCs.
TNF-, in the
presence of a fixed concentration of IFN- (1000 U/ml) stimulated
ET-1 release in a concentration-dependent manner with maximal
stimulation occurring at 10 ng/ml in both IMA (Fig.
1A) and SV (Fig. 1B) VSMCs. This
concentration of TNF- was selected for use in further experiments.
IFN- in the presence of a fixed concentration of TNF- (10 ng/ml)
similarly stimulated ET-1 release in a concentration-dependent manner,
with maximal stimulation occurring at 1000 U/ml in both IMA and SV
VSMCs (data not shown). As with TNF-, this concentration of IFN-
was selected for all further experiments. In other experiments,
addition of interleukin-1 (up to 10 ng/ml), interleukin-6 (up to 10 ng/ml), and bacterial endotoxin (up to 10 µg/ml) either separately or in combination produced no additional increase in the production of
ET-1 by either IMA or SV VSMCs.
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Time-Dependent Effect of TNF- and IFN- on Expression and
Production of ET-1 by Human IMA and SV VSMCs.
The combination of
TNF- (10 ng/ml) and IFN- (1000 U/ml) caused a sustained release
of ET-1 over 48 h from both IMA and SV VSMCs that was associated
with a very marked increase in expression of mRNA for prepro-ET-1, with
no changes in GAPDH (Fig. 2C).
Importantly, the increase in prepro-ET-1 mRNA preceded the elevation in
concentration of ET-1 within the culture medium (Fig. 2, A and B),
supporting the idea that (de novo) synthesis of ET-1 was occurring.
mRNA for prepro-ET-2 and prepro-ET-3 was undetectable.
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Effect of Phosphoramidon on Release of ET-1 and Big ET-1 from
Cytokine-Treated Human IMA and SV VSMCs.
The release of ET-1 that
followed exposure of the human VSMCs to TNF- and IFN- was
inhibited by phosphoramidon in a concentration-dependent manner (log
IC50 values: IMA, 3.9 ± 0.05; SV,
4.1 ± 0.09) with accompanying reciprocal increases in the
release of big ET-1 (log EC50 values: IMA,
4.4 ± 0.1; SV, 4.5 ± 0.09) (Fig.
3).
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Effects of CGS 26303, CGS 26393, and CGS 24592 on Release of ET-1
and Big ET-1 from Cytokine-Treated Human IMA and SV VSMCs.
CGS
26303 caused a concentration-dependent inhibition of the
cytokine-induced release of ET-1 from IMA and SV VSMCs with log IC50 values of 3.5 ± 0.09 and 3.6 ± 0.08, respectively (Fig. 4).
Similarly, CGS 26393 inhibited endogenous ET-1 release with a log
IC50 value of 4.4 ± 0.09 in IMA VSMCs and
of 3.8 ± 0.08 in SV VSMCs (Fig.
5). The inhibition of endogenous ET-1
production caused by both CGS 26303 and CGS 26393 was accompanied by
matched increases in the accumulation of big ET-1 (Figs. 4 and 5). The structurally related compound CGS 24592, a NEP inhibitor lacking in ECE
inhibitory activity, had no effect on cytokine-stimulated ET-1 release
from IMA or SV VSMCs (data not shown).
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Effect of Phosphoramidon and CGS 26303 on Conversion of Exogenous
Big ET-1 by Human IMA and SV VSMCs.
Phosphoramidon inhibited the
conversion of exogenous big ET-1 more potently than the endogenous
production of ET-1 in both human IMA VSMCs (log
IC50 values 4.8 ± 0.06 versus 3.9 ± 0.05, p < .0001) and SV VSMCs (log
IC50 values 5.1 ± 0.08 versus 4.1 ± 0.09, p < .0001; Fig.
6). Similarly, CGS 26303 inhibited the conversion of exogenous big ET-1 about 10 times more potently than the
endogenous production of ET-1 (log IC50 values:
IMA VSMCs, 4.3 ± 0.13 versus -3.5 ± 0.09, p < .001; SV VSMCs, 4.8 ± 0.15 versus
3.6 ± 0.08, p < .0001; Fig.
7).
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Effect of ECE Inhibitors on Cytokine-Stimulated Expression of
Prepro-ET-1 mRNA in Human VSMCs.
Phosphoramidon (300 µM), CGS
26303 (1 mM), or CGS 26393 (300 µM) had no effect on TNF- (10 ng/ml) and IFN- (1000 U/ml) stimulated expression of prepro-ET-1
mRNA over 48 h (Fig. 8).
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Expression of mRNA for ECE in Human SV VSMCs.
RT-PCR confirmed
the presence of ECE-1b/c mRNA in SV VSMCs (Fig.
9). No mRNA for ECE-1a was found in these
cells, although it was readily detected in human umbilical vein
endothelial cells used as a positive control. In contrast to the marked
elevation in prepro-ET-1, expression of mRNA for ECE-1b/c showed little variation during exposure of SV VSMCs to TNF- (10 ng/ml) and IFN-
(1000 U/ml) for 48 h, although a reduction was apparent at 48 h.
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Discussion |
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Top
Summary
Introduction
Experimental Procedures
Results
Discussion
References
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In this study we addressed two questions. First, can cytokines and/or endotoxin induce human VSMCs to produce ET-1, and second, how is endogenously produced big ET-1 processed?
As outlined in the Introduction, ET-1 appears to be involved
in many disease states associated with elevations in the plasma concentrations of proinflammatory cytokines. Because proinflammatory cytokines induce expression of other endothelial- derived mediators, such as NO and prostaglandins in smooth muscle, we were interested in
determining whether cytokines may also induce expression of ET-1. We
therefore examined the effects of interleukin-1, interleukin-6, TNF-, and IFN-, as well as bacterial endotoxin, on the production of ET-1 by human VSMCs. Combinations of TNF- and IFN- caused the
greatest increases in ET-1 production, with the other agents, either
alone or in combination, having no additional effect. Most notably, in
experiments examining the time course of the ET-1 response to TNF-
and IFN-, a very dramatic increase in ET-1 formation was evident
(Fig. 2). These experiments used large numbers of cells, permitting a
more reliable and precise sample analysis. The particular effects of
TNF- and IFN- in the human cells contrast with the rat in vivo,
in which we also found that interleukin-1 produces marked increases
in ET-1 production, albeit in a TNF--dependent manner (Klemm et al.,
1995). This draws attention to the variation in cytokine responsiveness
of vascular smooth muscle derived from different species, as has been
previously noted for the expression of iNOS (Beasley and McGuiggin,
1994). It is also important to note that the amount of ET-1 produced by
the human VSMCs after stimulation with cytokines (approximately 1 pg
per 103 cells in 48 h) is of a similar
magnitude to that of normal human aortic endothelial cells in culture
(our unpublished data, 1998). As in many blood vessels, VSMCs
form a much larger population than the single layer of endothelial
cells, production of ET-1 by the smooth muscle could clearly equal or
even surpass that by the endothelium.
After our experiments addressing the stimuli regulating ET-1 production
by VSMCs, we investigated the effects of known inhibitors of ECE.
Although ECE exists in two principal forms, ECE-1 and ECE-2, it is
ECE-1 that appears to be the enzyme mainly responsible for the
conversion of big ET-1 into ET-1 (Schmidt et al., 1994; Shimada et al.,
1994; Xu et al., 1994). It has been well documented that ECE-1 is a
zinc metalloprotease that is inhibited by phosphoramidon, and
phosphoramidon has been shown to inhibit the conversion of exogenous
big ET-1, both within the circulation and in isolated vascular
preparations (Ikegawa et al., 1991; Matsumura et al., 1991; McMahon et
al., 1991; Sawamura et al., 1991; Warner et al., 1992). Notably,
however, phosphoramidon is often more potent as an inhibitor of the
conversion of exogenous big ET-1 than as a suppresser of the endogenous
production of ET-1 by endothelial cells (McMahon et al., 1991; Sawamura
et al., 1991; Warner et al., 1992; Corder et al., 1993, 1995; Plumpton
et al., 1996). Similarly, we found that in the human VSMCs
phosphoramidon was 10 times more potent at inhibiting the conversion of
exogenous big ET-1 than the endogenous production of ET-1. This
difference was not confined to phosphoramidon, but was also seen with
CGS 26303, which displayed a similar 10-fold difference in potency (De
Lombaert et al., 1994; Trapani et al., 1995). Two pieces of evidence
suggest that the effects of phosphoramidon and CGS 26303 were mediated
at the level of ECE-1. First, and most important, the inhibition of
ET-1 production was accompanied by a reciprocal increase in the
accumulation of big ET-1 within the culture medium. Second, the
inhibitors of ET-1 production were not associated with reductions in
the expression of mRNA for prepro-ET-1. Third, CGS 24592, a NEP
inhibitor with little activity on ECE-1, had no effect on the
production of ET-1.
It is of particular interest that the ECE inhibitors acted with 10-fold
different potencies against the endogenous and exogenous conversion of
big ET-1 to ET-1. We can consider two likely reasons. First, that the
contrasting potencies are explained by the presence within the VSMCs of
multiple ECE enzymes differently responsible for the conversion of
endogenous and exogenous big ET-1, and second, that the conversion of
exogenous and endogenous big ET-1 may take place at different sites. To
address the first possibility, we used RT-PCR to assess the presence
within the VSMCs of ECE-1. There are three isoforms of ECE-1, ECE-1a,
ECE-1b, and ECE-1c that have distinct subcellular localizations
(Shimada et al., 1995; Valdenaire et al., 1995; Schweizer et al.,
1997). ECE-1a and ECE-1c are present at the cell surface, whereas
ECE-1b appears to be predominantly intracellular. We found no evidence
that the VSMCs express ECE-1a mRNA, although we could detect it in
control human umbilical vein endothelial cells, as previously reported (Schweizer et al., 1997). Conversely, RT-PCR revealed the presence of
ECE-1b/c in human IMA and SV VSMCs. These data indicate that the VSMCs
express known ECE-1 isoforms. Interestingly, we found no evidence that
the expression of the ECE-1b/c was increased following exposure to
cytokines, although small changes may well not have been detected by
the methodology we employed. This is in comparison with the clear
increase in expression of prepro-ET-1 that was detected in matched
experiments. It therefore appears that the second possibility
considered above is the most likely, i.e., VSMCs contain ECE-1b/c at
both intra- and extra-cellular sites. The intracellular ECE, possibly
ECE-1b, is responsible for processing endogenously produced big ET-1
and has a similar sensitivity to inhibitors of ECE, as does the
intracellular ECE-1b found in endothelial cells (Turner, 1993;
Schweizer et al., 1997). The resistance of this intracellular ECE-1b to
inhibition may be explained by inability of compounds to penetrate to
the inside of the cells. Phosphoramidon, for example, is a
phosphorylated sugar derivative and penetrates cells poorly (Turner,
1993; Corder et al., 1995). Earlier reports that vascular smooth muscle
converts big ET-1 to ET-1 in a readily inhibitable manner leading to
the conclusion that VSMCs cells express extracellular ECE, probably ECE-1c, is therefore too simple a conclusion (Balwierczak et al., 1993;
Plumpton et al., 1996; Maguire et al., 1997). In fact, VSMCs appear to
have an inducible synthetic pathway for ET-1 that is similar to that
present constitutively within endothelial cells, and similarly
sensitive as endothelial cell ECE to enzyme inhibitors.
In conclusion we show that cytokines can greatly increase the
expression and release of ET-1 by human VSMCs. Previous investigators have reported that VSMCs in culture can produce low amounts of ET-1,
and that this is increased following exposure to steroids, oxyhemoglobin, or cyclosporine (Kanse et al., 1991; Kasuya et al.,
1993; Takeda et al., 1993; Yu and Davenport, 1995; Schweizer et
al.,1997; Morin et al., 1998). It has also been found that VSMCs
cultured from coronary plaques produce more ET-1 than those cultured
from normal coronary artery (Haug et al., 1996). There has not,
however, been an analysis of the effects of proinflammatory cytokines
known to be involved in vascular disease states. Furthermore, there has
been no data presented as to the synthetic pathway via which ET-1 could
be produced. Generally, it has appeared that vascular smooth muscle is
a passive responder to ET-1, which expresses extracellular ECE to
convert any big ET-1 released from endothelial or other cell types. Our
data indicates that in fact the vascular smooth muscle can actively
produce ET-1 to a similar extent as the endothelium. The important
conclusion that we can draw from this is that the vascular smooth
muscle, traditionally thought to be an "ET-1 responder" can, under
the influence of cytokines, become an "ET-1 producer". This means
that we should add ET-1 to the list of vasoactive endothelial mediators
that can be induced in vascular smooth muscle: e.g., NO,
PGI2, PGE2, and now ET-1. It may well be that this is part of an adaptive response in which the
vascular smooth muscle takes on some endothelial characteristics to
compensate for endothelial dysfunction. Under such conditions, however,
the normal checks and balances present between mediators derived from
the endothelium, e.g., between NO and ET, may no longer be present
(Warner, 1996). This is indeed the case for NO, which can be produced
by iNOS in a largely unregulated manner (Wong and Billiar, 1995).
Unregulated production of ET-1 by the vascular smooth muscle following
loss or dysfunction of the endothelium may similarly underlie the
causative role of ET-1 in a number of disease states (Haynes and Webb,
1993; Warner et al., 1996; Parris and Webb, 1997).
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Footnotes |
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Received November 4, 1998; Accepted February 18, 1999
M. W. was in receipt of a European Commission Training and Mobility Fellowship, N. R. W. holds an Aelwyn Bursary from the Joint Research Board of St. Bartholomew's and the Royal London School of Medicine and Dentistry, J. A. M. is a Wellcome Foundation Career Development fellow, and T. D. W. is a British Heart Foundation Lecturer (BS/95003). Citation of meeting abstracts in which this work was previously presented: Woods M, Bishop-Bailey D, Pepper JR, Evans TW, Mitchell JA and Warner TD (1997) Cytokine and lipopolysaccharide stimulation of endothelin-1 release from internal mammary artery and saphenous vein smooth muscle cells. Br J Pharmacol 122:18P; and Woods M, Wood EG, Mitchell JA and Warner TD (1998) Evidence for intracellular endothelin converting enzyme in human vascular smooth muscle cells. FASEB J 12:A384.
Send reprint requests to: Dr. Timothy D. Warner, Vascular Inflammation, The William Harvey Research Institute, St. Bartholomew's and the Royal London School of Medicine and Dentistry, Charterhouse Square, London, EC1M 6BQ, United Kingdom. E-mail: t.d.warner{at}mds.qmw.ac.uk
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Abbreviations |
|---|
Big ET-1, big endothelin-1;
ECE, endothelin-converting enzyme;
ET-1, endothelin-1;
GAPDH, glyceraldehyde-3-phosphate dehydrogenase;
iNOS, inducible nitric oxide
synthase;
IFN-, interferon-;
IMA, internal mammary artery;
NEP, neutral endopeptidase;
NO, nitric oxide;
PGI2/E2, prostaglandin
I2/E2;
RT-PCR, reverse transcription-polymerase
chain reaction;
SV, saphenous vein;
TNF-, tumor necrosis factor-;
VSMCs, vascular smooth muscle cells.
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References |
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Top
Summary
Introduction
Experimental Procedures
Results
Discussion
References
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) and
production of ET-1 peptide (
) over time in VSMCs cultured from human
IMA (A) and SV (B). VSMCs were grown to confluence on
75-cm2 tissue culture flasks treated with a mixture of
TNF-
) and big ET-1 (
) in medium of IMA (A) and SV (B) VSMCs
treated with TNF-
) and big ET-1 (
) in medium of IMA (A) and SV (B) VSMCs
treated with TNF-
1 µM, 
