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National Cardiovascular Center Research Institute (Y.I., H.S., S.S., T.T., K.K., H.M., N.M.), Fujishirodai, Suita, Osaka 565, Japan; and Faculty of Pharmaceutical Sciences (Y.I., M.Y.), Setsunan University, Hirakata, Osaka 573–01, Japan
Address all correspondence and requests for reprints to: Naoto Minamino, Ph.D., National Cardiovascular Center Research Institute, 5-7-1 Fujishirodai, Suita, Osaka 565, Japan. E-mail: minamino{at}ri.ncvc.go.jp
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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inhibited AM secretion
from EC, whereas oxidized LDL stimulated it. Regulation of AM
production in EC is found to be similar to that of VSMC with several
exceptions, but AM and ET-1 production in EC are deduced to be
controlled independently and by different mechanisms. AM stimulates
cAMP production in EC, though receptors expressed on cultured rat EC
are not specific to AM but to calcitonin gene-related peptide. Based on
these findings, AM production in EC is thought to be regulated by a
variety of substances coming from blood and neighboring cells, and the
secreted AM is deduced to dilate blood vessels as an
endothelium-derived relaxing factor competing with ET-1. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Adrenomedullin (AM) is a potent vasorelaxant peptide of 52 residues recently isolated from human pheochromocytoma tissue (6). This peptide structurally belongs to the calcitonin-gene related peptide (CGRP) superfamily and elicits a potent depressor effect comparable to that of CGRP through direct action on vascular cells (7). Another bioactive peptide, PAMP (proadrenomedullin N-terminal 20 peptide), is generated from the N-terminal of the AM precursor protein and induces hypotensive activity through a distinct mechanism inhibiting norepinephrine release from sympathetic nerve endings (8, 9). We have demonstrated that EC and VSMC actively synthesize and secrete AM. Gene transcription levels of AM in cultured rat EC and VSMC are about 20 and 4 times higher than that of adrenal gland, and rat EC secretes AM at a rate comparable with that of ET-1 (10, 11). The secretion rate of AM from EC is about 5 times higher than that of VSMC, and immunoreactive (IR) AM secreted from EC and VSMC has been verified to be chromatographically and biologically indistinguishable from native AM. Furthermore, AM specific receptors coupled with an adenylate cyclase have been shown to be expressed on both EC and VSMC (12, 13, 14, 15). These findings suggest that AM secreted from EC plays an important role in the regulation of vascular tone.
We have reported that interleukin-1 (IL-1), tumor necrosis factor (TNF), and lipopolysaccharide (LPS), which are major factors inducing septic shock, most potently stimulate synthesis and secretion of AM from VSMC (11, 16). Glucocorticoid, thyroid hormone, and many vasoactive substances are found to alter AM synthesis, constituting a complex regulation system of AM production in VSMC (17, 18). Because ECs are recognized to have a critical function in the regulation of vascular tone through modulating and transmitting information coming from the blood stream to VSMC, we systematically surveyed substances stimulating and inhibiting AM production in cultured rat EC and compared them with those regulating AM production in VSMC and ET-1 production in EC.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(IL-1), IL-2, IL-3 and human TNF-ß
were obtained from Genzyme (Cambridge, MA), and mouse recombinant
IL-1ß from Intergen (Purchase, NY). Mouse recombinant TNF- and
bovine basic fibroblast growth factor (FGF) were products of Boehringer
Mannheim (Mannheim, Germany), and human recombinant epidermal growth
factor (EGF) was from Austral Biologicals (San Ramon, CA). Murine
recombinant interferon- (IFN-), IFN-ß and rat IFN- were
purchased from Calbiochem (San Diego, CA). Hydrocortisone,
testosterone, (-)-(R)-norepinephrine hydrogen tartrate
monohydrate, L-phenylephrine hydrochloride,
DL-isoproterenol hydrochloride,
12-O-tetradecanolyphorbol-13-acetate (TPA), forskolin,
heparin (sodium salt), and mannitol were obtained from Wako Pure
Chemicals (Osaka, Japan). Progesterone, 17ß-estradiol, Triton X-100
and Triton X-305 were products of Nacalai Tesque (Kyoto, Japan).
Escherichia coli LPS (serotype 026:B6) was purchased from
Paesel + Lorei (Frankfurt, Germany). Oxidized low density lipoprotein
(LDL) prepared by incubating LDL with EC was donated by Dr. Shimokado
of this institute, and lysophosphatidylcholine (lysoPC) was purchased
from Avanti Polar-Lipids (Alabaster, AL). NO generator, NOR-4
(3-{(±)-(E)-Ethyl-2'-[(E)-hydroxyimino]-5-nitro-3-hexenecarbamoyl}-pyridine),
was obtained from Dojindo Laboratories (Kumamoto, Japan), and
NG-nitro-L-arginine methyl ester
(L-NAME) was a product of Biomol Research Laboratory
(Plymouth Meeting, PA). Human AM(40–52) and its N-Tyr
derivative were synthesized by a peptide synthesizer (Applied
Biosystems, 431A) and purified by reverse phase HPLC. Human AM(22–52)
and human CGRP(8–37) were products of Peptide Institute (Osaka,
Japan). Other peptides were of rat origin and were obtained from
Peptide Institute, and rat CGRP-I was used as CGRP. All steroids were
first dissolved in ethanol, and then diluted with an incubation medium
(DMEM containing 0.01% BSA). T3 and reverse T3
were first dissolved in 0.1 M NaOH, and TPA was dissolved
in dimethylsulfoxide and then diluted with the incubation medium. Other
substances were dissolved according to the producer’s manuals and
diluted with the incubation medium.
Animals and preparation of aorta
The experiments were approved by the local committee on animal
experiments and care. Eight-week-old Sprague Dawley rats (Charles River
Japan, Yokohama, Japan) were maintained under normal conditions for at
least 1 week. LPS (5 mg/kg) or saline was injected through the tail
vein 30 min after ip administration of pentobarbital (25 mg/kg, Abbott
Laboratories, North Chicago, IL). At 3 h after injection of LPS,
subjects were injected ip with pentobarbital (50 mg/kg). The heart was
exposed, and cold saline containing heparin (5 U/ml) was infused
through the left ventricle. Then, the aorta was cut just above the
branching point of the renal artery and washed with saline. Whole
thoracic aorta was collected, washed well with HBSS, and associated
tissue was completely removed. For EC(-) tissue, ECs were swabbed off
with cotton buds after longitudinally dissecting the aorta. Total RNA
was extracted by the method described below.
Cell culture
Rat ECs were isolated from thoracic aorta by treatment with
0.25% trypsin, cloned, and maintained on collagen-coated dishes in
M199 medium containing 20% FCS (Hyclone, Logan, UT) and acidic FGF (5
ng/ml, Wako Pure Chemicals) of bovine brain origin at 37 C in a
humidified atmosphere containing 5% CO2 (10). The cloned
rat EC was identified by uptake of fluorescent acetylated LDL
(Biomedical Technologies, Stoughton, MA), active production of ET-1,
and negative immunostaining with monoclonal anti- smooth muscle
actin antiserum (Clone 1A4, Sigma). In this experiment, the ECs were
used at passage 11–20. Rat VSMCs were prepared by the explant and
enzyme dispersion method and maintained as reported previously
(11).
Preparation and processing of conditioned medium
ECs, grown to confluence in a collagen-coated 24-well plate
(Iwaki Glass, Tokyo, Japan), were washed twice with DMEM and incubated
in the incubation medium for 2 h. The media were then replaced
with incubation media containing the reagents to be tested, and
incubated at 37 C for 3–12 h in a CO2 incubator. After
being collected into polypropylene test tubes, the culture media were
acidified with acetic acid (final concentration, 0.25 M)
containing Triton X-100 (final concentration, 0.002%). The acidified
media were then heated at 100 C for 10 min, and lyophilized. The
lyophilizates were dissolved in a standard RIA buffer and
subjected to RIAs for AM and ET-1. Viability of EC after a 12-h
incubation was estimated by trypan blue exclusion assay, and more
than 98% of ECs were shown to be viable under the present conditions.
Longer incubation without FCS decreased viability of ECs and a
relatively high ratio of ECs are reported to start apoptosis after a
24-h incubation (19). On the other hand, we could not add FCS to the
incubation media because FCS and BSA altered AM production (Fig. 1
). For measurement of the effect of FCS,
BSA and mannitol, the incubation media were desalted and deproteinized
with Sep-pak C18 cartridge (Millipore Corp., Waters
Division, Millford, MA) (20).
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RIA for AM and ET-1
Details of the RIA system for AM using antiserum #172-CI-7
against human AM(40–52) have been reported previously (21).
N-Tyr derivative of human AM(40–52) was radioiodinated by
the lactoperoxidase method, and its N-Tyr-monoiodinated
form, isolated by reverse phase HPLC, was used as a tracer. Monoclonal
antibody against ET-1 was donated by Prof. Nakao (Kyoto University
School of Medicine) and RIA was carried out as reported (22).
RNA blot analysis
ECs, grown to confluence in a 10-cm collagen-coated dish, were
washed twice with DMEM and incubated with stimulants in the incubation
medium for 12 h. Total RNA was extracted by the acid guanidium
thiocyanate-phenol-chloroform method (10). Thoracic aortae collected
from LPS or saline injected rats were washed with HBSS. ECs were
removed by swabbing, washed well with the same solution, and then total
RNA was extracted from two aortae in each group by the same method.
After denaturation, RNA was electrophoresed and was then transferred to
Zeta probe membrane (Bio-Rad, Hercules, CA). Hybridization and washing
of the membrane were carried out as reported (10).
EcoRI-BglI complementary DNA (cDNA) fragment of
rat AM (nucleotide -153–422), PvuII cDNA fragment of rat
ET-1 (nucleotide 246–662), EcoRI-BamHI cDNA
fragment of rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
(nucleotide 492–799), and inducible nitric oxide synthase (iNOS) cDNA
fragment (nucleotide 2685–3345) were used as probes (20). Band
intensity was estimated by a Bioimage analyzer (BAS 5000, Fuji
Photofilm, Tokyo, Japan) and was compared after correction with GAPDH
messenger RNA (mRNA) as an internal standard.
Measurement of cAMP
ECs, grown to confluence in a collagen-coated 24-well plate,
were washed with DMEM and incubated in HEPES-buffered DMEM (pH 7.4)
containing 25 mM HEPES, 0.01% BSA, and 0.5 mM
isobutylmethylxanthine (Nacalai Tesque) for 1 h. The media were
then replaced with HEPES-buffered DMEM containing various
concentrations of AM, CGRP and their antagonists, and further incubated
at 37 C for another 1 h. Aliquots of the culture media were
succinylated, lyophilized, and then submitted for RIA of cAMP as
reported previously (23).
Receptor binding assay
ECs, grown to confluence in a collagen-coated 6-well plate, were
washed twice with 1 ml of 20 mM HEPES-buffered DMEM (pH
7.4), and incubated with 1.85 x 10-11 M
of 125I-labeled rat AM in 1 ml of DMEM containing 0.05%
BSA and 0.01% Triton X-305 at room temperature for 1 h.
Nonspecific binding was determined in the presence of 2
µM of unlabeled rat AM. After incubation, the cells were
washed once with ice-cold PBS and solubilized with 0.5 M
NaOH. Radioactivity was measured with a -counter (ARC-1000, Aloka,
Tokyo, Japan). Monoiodinated rat AM with biological activity was
prepared by the lactoperoxidase method and purified by reverse phase
HPLC.
Statistics
Values were expressed as mean ± SEM.
Statistical analysis of the results was performed with a one-way ANOVA,
followed by a multiple comparison test (Dunnett’s test), and
P < 0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Based on data of regulation of AM synthesis and secretion from cultured
rat VSMC (16, 17, 18), we subjected 43 substances to the AM production
assay of cultured rat EC. We simultaneously monitored concentrations of
IR-ET-1 in the culture medium, which was also synthesized and
constitutively secreted from EC. As shown in Table 1, 26 out of 43 substances significantly
influenced IR-AM content in the medium of ECs after 12-h incubation,
i.e. 19 substances increased and 7 substances decreased AM
secretion. Interestingly, BSA was found to be one of the most
stimulatory reagents, whereas FCS was one of the most inhibitory
substances of AM secretion from cultured rat EC, as shown in Fig. 1
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BSA dose dependently stimulated AM secretion, and the secreted IR-AM
level was 2.4 times higher than that of the control. BSA elevated
IR-ET-1 content in the medium in low concentrations but strongly
reduced it at concentrations higher than 0.25%. We examined the
effects of BSA up to 2.5%, but 2.5% BSA induced a stimulatory effect
on AM secretion only a little stronger than that of 0.5% BSA. To check
whether the effect of BSA on AM and ET-1 secretion from EC was derived
from osmotic pressure, mannitol was added to the medium. Although we
dissolved mannitol up to a concentration corresponding to 5% BSA, it
did not affect secretion levels of AM and ET-1 at all. In contrast with
BSA, FCS lowered IR-AM content in the medium to 55% and increased
IR-ET-1 content up to 132%. Therefore, we employed a serum-free
culture medium containing a low concentration (0.01%) of BSA of the
same preparation in this study to avoid interference of FCS and
BSA.
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, IL-1ß, TNF-, TNF-ß,
and LPS augmented AM secretion from rat EC, as in the case of rat VSMC.
IL-1 and TNF- increased IR-AM content in the culture medium about
2.2-fold, and IL-1ß, TNF-ß, and LPS elevated it but to a lesser
extent. However, these cytokines and LPS did not significantly alter
IR-ET-1 content. As shown in Fig. 2, IL-1ß did not increase IR-AM content in the medium at concentrations
lower than 0.02 ng/ml but elevated it at concentrations higher than 0.2
ng/ml. LPS induced a significant increase of IR-AM content only at a
dose of 20 ng/ml. TNF- dose dependently augmented IR-AM content in
the medium as well as mRNA levels in the cells, as shown in Fig. 3A. TNF-ß also increased IR-AM content
in the medium, but not as dose dependently as TNF-. IL-1
significantly elevated the IR-AM level at concentrations between 0.02
and 0.2 ng/ml but did not affect it at other concentrations.
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. Basic FGF and EGF showed weak but significant
inhibitory effects on AM secretion but no effect on ET-1 secretion.
Among the three IFNs, only IFN- showed a weak inhibitory effect,
decreasing IR-AM content to 80% of control, and none of the IFNs
altered IR-ET-1 content.
Thrombin potently elevated both AM and ET-1 concentrations in the
medium up to 283% and 155% of the control, respectively. However, no
apparent change was observed in the mRNA band intensity of AM and ET-1
after a 12-h stimulation with thrombin (Fig. 3C).
We checked the effects of six steroid hormones, including glucocorticoid, mineralocorticoid, and sex steroids, on AM secretion from EC. All of these steroid hormones increased IR-AM contents in the medium of ECs, although they produced, at most, a 35% elevation compared with the control. On ET-1 secretion, only DEX and hydrocortisone showed significant effects. Thyroid hormone, T3, increased both IR-AM and IR-ET-1 contents in the medium by about 20%, and its inactive isomer, reverse T3, did not elicit a significant effect on either IR-AM or IR-ET-1 level.
In the four catecholamines examined, isoproterenol and norepinephrine slightly but significantly increased IR-AM content in the medium, but none of these catecholamines altered IR-ET-1 content. We also administered six vasoactive peptides, vasoactive intestinal polypeptide (VIP), CGRP, ET-1, atrial natriuretic peptide (ANP), angiotensin II, and substance P, to the medium of EC. CGRP and ET-1 decreased the IR-AM level in the media to about 85% of the control, and VIP decreased IR-ET-1 level to 85% of the control.
NO synthase inhibitor (L-NAME) and NO generator (NOR-4) did not alter IR-AM or IR-ET-1 content in culture medium of rat ECs. On the other hand, oxidized LDL and lysoPC increased IR-AM content in the medium about 55% and 22%, whereas these substances did not elicit a significant effect on IR-ET-1 content.
As shown in Fig. 2C, TPA elevated both IR-AM and IR-ET-1 contents in
the culture medium of rat ECs in lower concentrations,
10-10–10-9 M for AM and
10-12–10-11 M for ET-1. At
concentrations higher than 10-8 M, TPA weakly
reduced IR-AM content and markedly lowered IR-ET-1 level. We
administered forskolin and 8-Br-cAMP to ECs up to 10-5
M and 10-3 M, respectively, but no
significant alteration was observed in IR-AM or IR-ET content in the
medium. 8-Br-cGMP did not show any effect on either AM or ET-1
secretion.
We checked the cooperativity of TNF-, IL-1ß, and LPS on AM
synthesis and secretion from cultured rat EC (Fig. 4) because these substances often induce
a synergistic effect when added simultaneously. Each of the three
substances administered alone increased IR-AM content in the culture
medium. Coadministration of IL-1ß and LPS or TNF- and LPS almost
additively elevated IR-AM content in the culture medium. However,
administration of IL-1ß with TNF- or with TNF- and LPS reduced
the increase in IR-AM content in the medium.
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at doses inducing maximal
effects on AM secretion (data not shown). DEX induced a 2.4-fold
increase in the AM mRNA level compared with the control, and IFN-
slightly enhanced AM mRNA band intensity. In the case of ET-1 mRNA, DEX
and IFN- produced relatively large increases (3.2-fold and 2.2-fold)
in the mRNA band intensity. These results, as well as those shown Table 1 and Fig. 3, indicate that the level of AM secreted from EC is not
well correlated with the AM mRNA level as observed in the case of VSMC
(11, 16, 17, 18).
Next, we examined properties of AM receptors expressed on the cultured
rat EC used for the present study. As shown in Fig. 5, rat CGRP dose dependently elevated cAMP production with an
ED50 value of 1.2 x 10-10 M,
and AM antagonist, human AM(22–52), did not affect the dose-response
curve. In the presence of 10-6 M of CGRP
antagonist, human CGRP(8–37), the dose-response curve of CGRP was
markedly shifted to the high concentration side and the
ED50 value was 200 times higher than that without CGRP
antagonist. In contrast, the ED50 value of rat AM in the
cAMP production was 5.3 x 10-8 M, being
440 times higher than that of rat CGRP. The dose-response curve of AM
was not shifted by addition of human AM(22–52) but was shifted with
human CGRP(8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37). We also checked two other lines of rat EC, both of
which showed higher affinity for CGRP than AM, as in the case of Fig. 5. We also tried to characterize AM receptors by receptor binding assay
using a radiolabeled rat AM purified by reverse phase HPLC. No specific
binding of AM to cultured rat EC was observed, although we found
significant binding of the ligand and a clear displacement curve in the
case of bovine EC under the same conditions used in the present
study.
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). At 3 h
after injection of LPS (5 mg/kg), the AM mRNA level and iNOS mRNA level
in the whole aorta were increased 4-fold and 8-fold compared with that
of saline-injected control rats. By removal of ECs by swabbing, the AM
mRNA level was reduced to 80% of the nontreated aorta, whereas that of
iNOS, which was mainly transcribed in VSMC, was increased to 120%.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). This result indicates
that AM gene transcription in EC significantly contributes to the total
gene transcription in the thoracic aorta, although the mass of EC is
less than a few percent of the aorta. Based on these findings, EC
should be recognized as a major source of AM even in the in
vivo system. In this study, we measured IR-AM content in the culture medium of ECs after a 12-h incubation with various substances as an index of AM synthesis. To elucidate physiological functions of AM in the vascular wall, it is necessary to know how AM production in EC is regulated, even though AM content in the medium is not always altered by direct action of the administered substance during a 12-h incubation. We also compared the regulation of AM production in EC to that in VSMC as well as to the regulation of ET-1 production in EC to help elucidate the functions of AM secreted from EC.
IL-1s, TNFs, and LPS, which are known to be major factors inducing
septic shock, significantly increased AM mRNA levels and AM secretion
from EC (Figs. 2 and 3), although not as potently as in VSMC (16). ET-1
secretion was not altered by these inflammatory cytokines or by LPS.
Coadministration of TNF- and LPS or IL-1ß and LPS additively
increased AM content in the culture medium of rat ECs, but the effect
of TNF- was reduced in the presence of IL-1ß (Fig. 4). This result
indicates that AM synthesis in EC stimulated with LPS is augmented by
the addition of TNF- or IL-1ß, although their cooperativity in EC
is not as high as that observed in VSMC (16). Thus, AM synthesis in the
vascular wall is augmented with TNF, IL-1 and LPS, and the secreted AM
appears to bind receptors on VSMC and effectively dilate blood
vessels.
We have demonstrated that plasma concentration and gene transcription of AM are highly elevated in LPS-injected rat (20). We have also found that plasma AM concentration in patients with septic shock is markedly increased and correlates negatively with systemic vascular resistance index (25). Hirata et al. also reported high levels of plasma AM in septic patients (26). These findings, along with the in vitro data obtained from cultured rat EC and VSMC, indicate that an increase in plasma AM concentration in LPS-injected rats and patients with septic shock results from augmented synthesis and secretion of AM from EC and VSMC. NO is recognized as a major substance inducing hypotension in septic shock (27, 28), but the results mentioned above support the hypothesis that AM generated from EC and VSMC participates in the refractory vasodilation of sepsis.
As shown in Fig. 2C, TPA elicits dual regulation of AM synthesis,
i.e. stimulation at lower and inhibition at higher
concentrations. Because long exposure to a high concentration of TPA is
known to inhibit protein kinase C through a feedback mechanism (29),
its dual effects are thought to be induced through the protein kinase C
pathway. AM synthesis in rat VSMC is also stimulated at low
concentrations of TPA, and AP-1 and AP-2 sites are present in the
5'-regulatory region of human AM gene (30). The protein kinase C
pathway participates in the regulation of AM gene transcription to some
extent. On the other hand, forskolin and cAMP analog failed to alter AM
synthesis in EC, in contrast to VSMC where these reagents suppressed AM
synthesis (18). Although cAMP responsive element is present in the
5'-upstream region of human AM gene (30), the cAMP-mediated pathway may
not contribute to the activating factor for AM gene transcription in
the vascular wall cells. CGRP, which also increased intracellular cAMP
production, weakly suppressed AM synthesis. This result suggests that
the suppressive effect of CGRP is not caused simply by the
cAMP-mediated pathway.
TNF-, IL-1-, and LPS-induced activation of gene transcription is
considered to be mainly mediated by transcriptional factor NF-
B
(31). In the 5'-upstream region of human AM gene, there are several
sites that differ from the 10-base consensus sequence of NF-B
binding site by one base replacement (30, 32). In the case of VSMC,
TNF, IL-1, and LPS exert their effect additively, suggesting that these
three substances activate AM gene transcription through the same
pathway. TNF--induced AM synthesis is suppressed by IL-1ß in EC,
although LPS showed cooperative effects with both TNF- and IL-1ß
(Fig. 4). Because TNF--induced gene transcription is reported to be
inhibited by IL-1 in several proteins of rat and human EC (33), AM
synthesis stimulated with IL-1ß and TNF- in EC might be mediated
through different pathways. We administered several different
inhibitors of the signal transduction, such as H-7, to identify the
pathways used for activation of AM gene transcription stimulated with
TPA and cytokines but were not able to observe significant effects on
AM content in the medium probably due to cytotoxicity of the
inhibitors.
TGF-ß1 was found to be the most potent suppressor of AM
synthesis, whereas it is the strongest stimulator of ET-1 synthesis
(Fig. 3B, Table 1). TGF-ß1 has also been reported to
increase production of EC-derived vasodilators, prostacyclin and C-type
natriuretic peptide (34, 35). These findings indicate that
TGF-ß1 is a factor regulating not only growth and
differentiation of vascular cells but also vascular tone through
alteration of production of vasoactive substances. Because
TGF-ß1 is a potent stimulator of ET-1 synthesis and
secretion, it is possible that the secreted ET-1 secondarily elevates
AM synthesis in EC. However, the concentration of ET-1 was 2 x
10-10 M after a 12-h incubation, which
was too low to stimulate AM synthesis.
FCS potently decreased AM content and increased ET-1 content in the
culture medium of ECs in a manner similar to that of
TGF-ß1 (Fig. 1), and no other substances induced
comparable effects. Although plasma TGF-ß1 concentration
(ca. 0.1 ng/ml) is not sufficient to induce an inhibitory effect, a
latent form of TGF-ß1 is expressed on EC (36, 37).
Because plasminogen in FCS is activated by plasminogen activator
secreted from EC, the activated plasmin can process the latent form to
mature TGF-ß1 (37). These findings suggest that the
suppression of AM synthesis with FCS is induced by TGF-ß1
generated under the culture conditions. The strong inhibitory effect of
FCS partly explains why AM gene transcription is highly suppressed in
the in vivo conditions observed in intact aorta (10). On the
other hand, no substance has been found to elicit effects comparable to
BSA that markedly elevated AM content and reduced ET-1 content in the
medium of EC. We also administered higher concentrations of BSA, up to
2.5%, which elicited effects comparable to or a little stronger than
those observed for 0.5% BSA. On the other hand, osmotic stress induced
with mannitol, which corresponded to 5% BSA, did not alter AM
secretion from EC at all. Because serum albumin is reported to inhibit
prostacyclin synthesis in EC (38), it is necessary to determine whether
the effects are induced by BSA itself or by another associated
substance which may alter synthesis of vasoactive peptides.
EGF and basic FGF weakly suppressed AM secretion (Table 1). But their
effects might be indirect because lower doses of these substances do
not induce any effect. IFN- showed a suppressive effect on secretion
of AM from both EC and VSMC. In the 5'- upstream region of human AM
gene, there are many -IFN responsive elements (30), but these do not
affect AM gene transcription in either EC or VSMC, in contrast with
iNOS gene which has -IFN responsive elements and is highly activated
with IFN- in the presence of LPS (39). Thrombin is a potent
stimulant of AM and ET-1 synthesis in EC. This is in sharp contrast to
VSMC, where thrombin most potently inhibits AM synthesis (18). Thrombin
is known to work as a proinflammatory agent by promoting the synthesis
of IL-8 and other cytokines (40). Stimulatory effects of thrombin on AM
and ET-1 synthesis in EC may be induced not as specific but general
reactions of EC against thrombin. Thrombin acts on EC and elicits its
effect through the EC-mediated pathway in the normal vascular wall, but
it acts on VSMC when the vascular wall is wounded. Thus, the resulting
effect of thrombin on AM production in the vascular wall is deduced to
be different between normal and wounded blood vessels.
All steroid hormones weakly stimulate AM secretion from EC, although glucocorticoid specifically augments it from VSMC (17). Based on their nonspecific effects, steroid hormones might influence AM synthesis in EC by secondary reaction. In contrast, thyroid hormone is found to increase synthesis and secretion of AM and ET-1 through specific receptors. Imai et al. (41) reported specific and potent effects of glucocorticoid on AM gene transcription in EC but observed no specific stimulation of steroid hormones in this study. Because isoproterenol and norepinephrine slightly stimulated AM secretion, their effects may be mediated by ß-receptor.
In the vasoactive peptides, ET-1 weakly suppressed secretion of AM from EC. Suppression of AM secretion by ET-1 might strengthen the effect of ET-1 secreted from EC. ANP did not alter AM and ET-1 synthesis at all. Vesely et al. (42) reported that ANP infusion elevated plasma AM concentration within 20 min in healthy human subjects, but its time dependency was quite different from that observed when AM synthesis in EC and VSMC was stimulated (16, 17). Their results suggest that AM stored in the endocrine-type cells is secreted into the blood by ANP infusion. Based on the results of regulation of AM synthesis and secretion, EC and VSMC cannot be the source of the transient increase of plasma AM. Gene transcription of iNOS and nitrite production have recently been reported to be stimulated by AM (43, 44). We administered an NO synthase inhibitor and generator to EC vice versa, but no significant effect was observed on basal AM nor on ET-1 production in rat EC. As for ET-1 production in EC, Cao et al. (45) reported that L-NAME increased ET-1 secretion from human umbilical vein EC (45), but we observed no stimulatory effect of L-NAME on ET-1 secretion from human aortic EC (our unpublished observation).
Oxidized LDL increased AM secretion 58%, which was lower than the
effect of IL-1 but higher than that of IL-1ß. LysoPC, a
phospholipid fraction of LDL, also stimulated AM secretion. Oxidized
LDL is a major substance inducing atherosclerosis and stimulates
secretion of growth factors and cytokines that proliferate VSMCs (46).
PDGF- or FCS-stimulated growth and migration of VSMC were reported to
be inhibited by AM (47, 48), and we also obtained similar results (our
unpublished observation). AM secreted from EC by the stimulation of
oxidized LDL and cytokines may exert antiproliferative effects on VSMC
in the atherosclerotic region.
When regulation of AM production in EC is compared with that in VSMC,
most of the substances influence AM production in the same direction,
even though their relative potencies are smaller in EC. For example,
substances inducing septic shock, such as IL-1, TNF and LPS, potently
stimulate AM synthesis and secretion from both EC and VSMC. Steroid and
thyroid hormones as well as phorbol ester increase AM synthesis in EC
and VSMC, whereas IFN- suppresses it. Typical differences in AM
synthesis between EC and VSMC are induced by FCS, thrombin, FGF, EGF,
and ET-1. FCS as well as FGF, EGF and ET-1 stimulate AM synthesis in
VSMCs and inhibit it in ECs, whereas thrombin enhances AM synthesis in
ECs and reduces it in VSMCs. Because these substances have
proliferative effects on VSMCs in addition to regulating the production
of vasoactive peptides, their activity should be evaluated based on
short-term vascular tone regulation as well as long term vascular cell
growth. The difference in regulation of AM production between EC and
VSMC provides a clue to elucidating functions of AM secreted from EC
and VSMC.
Among substances which significantly altered either AM or ET-1 synthesis in EC, only thrombin, thyroid hormone and TPA stimulated both AM and ET-1 synthesis. Thyroid hormone receptor-mediated pathway and protein kinase C pathway are probably shared with AM and ET-1 in rat EC. Most of the other substances induce distinct effects on AM and ET-1 synthesis in EC, as typically observed in TGF-ß1 which stimulates ET-1 synthesis but suppresses AM synthesis. Based on these data, AM and ET-1 synthesis in EC is deduced to be generally regulated by independent and different mechanisms. The two vasoactive peptides with opposing biological activity are deduced to be produced in EC by distinct regulation and to participate in the control of vascular tone as competitors.
AM-specific receptors are shown to be expressed on cultured human and
bovine ECs (12, 13). As shown in Fig. 5, however, presumed AM receptor
on rat EC is found to be specific to CGRP but not to AM on the basis of
its affinity and behavior to antagonists. Several different rat ECs,
which actively synthesize and secrete AM and ET-1, also showed high
affinity for CGRP. Because cAMP producing properties of EC receptors
are quite similar to those reported for CGRP type-I receptor (49), CGRP
receptor is concluded to be a major receptor expressed on cultured rat
EC. If this is true in the intact blood vessel, AM is able to function
as an EC-derived vasorelaxant but not as an autocrine regulator of EC
in rat.
In conclusion, AM production in rat EC is regulated by many cytokines, hormones, and vasoactive substances. Most of the substances regulate AM production in EC and VSMC in the same direction, but AM production and ET-1 production in EC are regulated independently and differently. ECs significantly contribute to total AM production in the vascular wall even in the aorta, and secreted AM participates in the regulation of vascular tone as an endothelium-derived relaxing factor competing with ET-1.
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Acknowledgments |
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Footnotes |
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Received July 7, 1997.
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References |
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