© 1997 American Heart Association, Inc.
Articles |
Response of Vascular Smooth Muscle Cells to the Neuropeptide Secretoneurin
A Functional Role for Migration and Proliferation in Vitro
From the Department of Internal Medicine, Faculty of Medicine, University of Innsbruck, Innsbruck, Austria.
Correspondence to Univ. Prof. Dr. C. J. Wiedermann, Department of Internal Medicine/Intensive Care Medicine, University of Innsbruck, Anichstrasse 35, 6020 Innsbruck, Austria.
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Abstract Mesenchymal cell migration and replication are central biologic events involved in atherosclerosis and lung and hepatic fibrosis. Tissue repair and fibrosis are thought to be regulated by growth regulatory molecules, comprising both stimulators and inhibitors of mesenchymal cell functions, including platelet-derived growth factor (PDGF), transforming growth factor-ß (TGF-ß), fibroblast growth factors, and several neuropeptides such as substance P. Secretoneurin (SN), a novel 33-amino acid neuropeptide derived from secretogranin II (chromogranin C), is widely distributed in the central and peripheral nervous and neuroendocrine systems, including afferent C-fibers, and can be released in the periphery by capsaicin. Recently, we reported that SN triggers the selective migration of human monocytes and fibroblasts in vitro, implicating its involvement in inflammatory responses. We report herein that SN stimulates specific migration (maximal response at 10-10 M) of cultured arterial smooth muscle cells (SMCs), originating from rat thoracic aorta, and initiates DNA synthesis and SMC growth (BrdU incorporation, MTT test) with a maximum at 10-8 M SN to a similar extent as observed by PDGF. Both functional activities of SN were inhibited by specific anti-SN immunoglobulins (dilution, 1:1000), and furthermore, a trypsinized SN peptide (10-8 M) was unable to provoke biologic effects. Our studies suggest that SN functions as a regulatory peptide to modulate SMC migration and proliferation, which in conjunction with other factors could serve to aggravate and accelerate the development of atherosclerotic or restenotic lesions at sites of vascular injury.
Key Words: secretoneurin • smooth muscle cells • migration • proliferation
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Introduction |
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The walls of blood vessels consist to a large extent of vascular smooth muscle cells and their extracellular matrix products.1 In comparison with smooth muscle cells (SMCs), endothelial cells and adventitial fibroblasts each represent a small proportion of the total number of cells found in the vascular wall.2 Atherosclerosis is a disease of the intima and primarily involves different cell types, i.e., endothelial and smooth muscle cells, monocytes, T-lymphocytes, and platelets, causing expanding intramural plaques that thicken the arterial wall and encroach on the vascular lumen, thus giving rise to reduced blood flow, ischemia, and infarction. Its pathobiology is believed to represent an abnormal expression of the processes involved in vascular healing. Etiologic models derive from a "response-to-injury" paradigm that can be divided in three separate disease stages: endothelial dysfunction, smooth muscle cell activation, and architectural disruption. Migration and proliferation of medial SMCs occur not only in the pathogenesis of atherosclerotic lesions but also in restenosis after vascular interventions.3 4 5 6 7 8 Both migration and proliferation result from the binding of chemotactic and trophic factors to SMC receptors, which initiates a cascade of intracellular molecular events leading either to cytoskeletal locomotory restructuring or to cell cycle activation. Platelet-derived growth factor (PDGF) is a powerful chemoattractant for SMCs9 and induces SMC replication.10 PDGF is secreted among several other cell types by activated platelets and macrophages at sites of inflammation and vascular damage. Proliferation of quiescent SMCs is stimulated within hours of exposure to PDGF.10 11 Similarly, SMC migration to PDGF has been demonstrated in vitro and in vivo. In addition to PDGF, a large number of growth factors, cytokines, and vasoregulatory peptides, such as endothelium-derived growth factor12 13 or SMC-derived PDGF-like growth factor,14 participate in this inflammatory-fibroproliferative process.
SMCs, fibroblasts, and the endothelium are major targets of the neuroendocrine system and are capable of expressing a variety of functional receptors for neurotransmitters and neuromodulators in vivo. Neurohumoral substances can affect vascular tone15 and were shown to modulate endothelial and mesenchymal functions, such as migration and proliferation, e.g., substance P.16 17 18 19 In addition, experimental data have implicated substance P's role as a growth20 and chemotactic21 factor for cultured endothelium. Secretoneurin (SN), a recently discovered 33-amino acid-long secretogranin II-derived neuropeptide, was reported to act in a manner typical of sensory neuropeptides.22 23 24 Secretogranin II is a member of the chromogranin family, the acidic proteins of secretory granules.25 SN is co-released with other neuropeptides, e.g., substance P from afferent C-fibers in the periphery by capsaicin.26 It seems to act as a proinflammatory protein, regulating migration and proliferation of cells involved in inflammatory responses. Thus, SN stimulates chemotactic migration of monocytes, but not of polymorphonuclear leukocytes,27 and stimulates migration of human fibroblasts in vitro.28
As the inappropriate migration and proliferation of vascular SMCs within the intima of vessel walls are precipitating factors in the development of atherosclerotic lesions,4 we investigated the effect of SN on these SMC functions. Our results showed that SN stimulates DNA synthesis and an increase in the cell number of SMCs by specific mechanisms to an extent similar to PDGF. Moreover, we report herein that SN acts as a potent chemoattractant for cultured SMCs in vitro.
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Materials |
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Purification of Peptides and Production of Antisera
Five peptides derived from the primary amino acid sequence of secretogranin II were kindly provided by the Department of Pharmacology, University of Innsbruck, as described previously:23 bovine secretogranin II, 133-151 (bLF-19); bovine secretogranin II, 270-286 (bEL-17); rat secretogranin II, 154-186 (rSN); human secretogranin II, 152-184 (hSN); human secretogranin II, 172-186 (carboxyl-terminal fragment of SN); and a trypsinized SN peptide. Proteolytic cleavage of secretoneurin was done by trypsin (350 µg of secretoneurin in 80 µl of 0.1 M Tris-HCl, pH 7.9, containing 10 µg of trypsin for 3 hours at 37°C). Purity of peptides was verified by amino acid sequencing and mass spectrography, and the concentrations were determined by amino acid analysis based on phenylalanine. Contamination by endotoxin was determined by E-TOXATE assay (Limulus amebocyte lysate, Sigma Chemical Co., St. Louis, Mo). For antisera production, SN was coupled via an additional amino-terminal cystein to maleimide-activated keyhole limpet hemocyanin and used for generation of antisera in Chincilla Bastard rabbits (Ivanovas, Kislegg, Germany) as previously reported in detail.22 Immunoglobulin fractions were prepared from antiserum by affinity chromatography on Protein A Sepharose columns (Pharmacia, Bromma, Sweden).
Conditions of Smooth Muscle Cell Cultures
SMCs, which were originally isolated from rat thoracic aorta of
BDIX rats (A7r5), were obtained from the American Type Culture
Collection, Rockville, MD. SMCs were grown in enriched medium
(Dulbecco's modified Eagle medium (DMEM; Biological Industries, Beth
Haemek, Israel) supplemented with L-glutamine, 10% fetal
calf serum (FCS), 100 U/mL penicillin, and 100 µg/mL
streptomycin) in 75 cm2 plastic tissue culture flasks and
kept in a humidified incubator at 37°C in 5% CO2. After
reaching confluence, the cells were subcultured and reseeded at a ratio
of 1:3. Smooth muscle cells were used between passages 3 and 10 for
all experiments.
Proliferation Assays
The effects of SN on SMC growth were monitored by DNA synthesis
using BrdU incorporation, and the increase in cell number was measured
by MTT assay.29
Preparation of Smooth Muscle Cells
Replicate confluent SMC cultures were washed and trypsinized,
and 100 µL of cell suspension (5x103 cells/well) in DMEM
containing 10% FCS was plated into 96-well flat-bottomed tissue
culture plates and used for experiments 24 hours after trypsinization.
Before initiation of growth studies, the cells in exponential growth
were then switched into DMEM supplemented with 0.2% FCS for 48
hours.30 This incubation under serum-starved conditions
deprives the cells of serum-associated mitogens, arrests growth, and
synchronizes cell proliferation in response to proteins subsequently
assayed. Proliferation assays were performed with SMCs growing in
medium supplemented with 0.5% FCS (basal conditions) and with 10% FCS
(optimal growth conditions) as a positive control.
Colorimetric Assay for Cellular Growth and Survival
(MTT assay)
After incubation at 37°C for the various time periods in
medium containing assay substances, cellular proliferation was measured
using a colorimetric assay for cell growth and
chemosensitivity.31 This calorimetric assay based on the
tetrazolium salt MTT ((3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyl
tetrazolium bromide; Sigma Chemical Co) detects living but not dead
cells, and the signal generated is directly proportional to the number
of cells.29 As previously described, stock MTT solution
was added to all wells of an assay plate.29 31 After a
further period of incubation (6 hours), the medium was aspirated from
the adherent cells as completely as possible without disturbing the
formazan crystals formed within the cells. Subsequently,
dimethylsulfoxide (Merck, Darmstadt, Germany) was added to each well,
the plates were agitated on a plate shaker, and the optical density was
read. Optical density measurements were carried out with an
enzyme-linked immunosorbent assay reader (model MR 700; Dynatech Labs.,
Guernsey, United Kingdom) at 570 nm.
BrdU Incorporation Assay
DNA synthesis in SMCs was measured using the
5-bromo-2'-deoxy-uridine (BrdU)-Labeling and Detection Kit (BrdU-Kit
III, Boehringer Mannheim, Ingelsheim, Germany). BrdU was added
to the culture medium for incorporation into the DNA of replicating
cells. After 2 hours of incubation, cells were fixed and nucleases
added to partially digest and expose the target haptens. Thereafter,
anti-BrdU-peroxidase was added to each well to bind to BrdU, and
finally the signal, developed with a water-soluble peroxidase substrate
enhanced by a substrate enhancer (1 mg/mL), was measured with
the enzyme-linked immunosorbent assay reader. Cell viability after BrdU
treatment was tested by trypan dye exclusion in preliminary
experiments. Cell viability was always >98%. Measurement of
extinction of the samples was carried out in a microliter plate reader
at 405 nm with a reference wavelength at 490 nm. In any given
experiments for a given condition, the variation between wells of the
percentage of cells labeled was <5%.
Migration Assays
Preparation of Smooth Muscle Cells
Trypsinized SMCs were resuspended in complete medium, reseeded
at 1.5x106 cells in Petri dishes, and starved in 0.2%
FCS-containing medium for 48 hours. Cells were trypsinized with 0.05%
trypsin and 0.02% EDTA at 37°C until they were observed to round up,
which typically occurred after approximately 1 minute. DMEM containing
20% FCS was then added to the cultures to stop trypsinization. After
centrifugation, cells were washed twice and resuspended
in DMEM supplemented with 0.5% BSA (Behring, Marburg, Germany) at
1x106/mL and allowed to recover in suspension for 30
minutes. The cells were then finally centrifuged, resuspended,
and used for migration experiments.
Transwell Assay
Cell migration assays were performed as previously
described32 33 34 using modified Boyden chambers with a
6.5-mm, 8-µm pore size,35 36 37 10-µm thickness
polystyrene membrane separating the two chambers (Transwells,
Costar, NL). Assay substances dissolved in medium containing 0.5% BSA
were placed into the lower chamber of a 24-well plate containing
Transwells and allowed to stand at 37°C for 30 minutes before
placement of the cells into the insert (1x105 cells/well).
Transwell inserts were pretreated with human fibronectin (10
µg/mL; Sigma Chemical Co). Cell migration was measured after 4
hours of incubation at 37°C.34 All nonmigrant cells were
removed from the upper face of the Transwell membrane with a cotton
swab, and migrant cells, i.e., those attached to the lower face, were
fixed with methanol and stained with Diff Quick (Dade, Düdingen,
Switzerland). Stained cells were subsequently extracted with 10%
acetic acid, and the absorbance was determined at 600 nm. In all
experiments, we used PDGF AB (10 ng/ml) as positive chemotactic
control34 and medium supplemented with 0.5% BSA as
negative control to determine random cell migration.
Calculations
The data are expressed as mean±SEM. The
nonparametric analysis was performed for
independent samples after Kruskal-Wallis. Differences were compared
using the Mann-Whitney U test or the two-tailed Student's
t-test for paired samples.
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Secretoneurin Stimulates SMC Migration
The ability of SMCs to migrate in response to soluble SN over a broad concentration range (10-14 to 10-6 M) was observed in the migration assays. Results showed a dose-dependent migration to SN in the range from 10-12 to 10-6 M. A maximal response was observed at approximately 10-10 M of SN (Fig 1
), and
the number of migrated cells was approximately 115% above the basal
level (random migration). Direction of migration (chemotaxis) was
established by comparing the response of SMCs with SN being added to
either the upper or lower compartments; to distinguish between
chemotaxis and chemokinesis, the same concentration of chemoattractant
was incorporated into both the lower and upper compartments of each
well (checkerboard analysis). No significant stimulation of
migration was observed toward negative gradients of SN (data not
shown). Similar to previous observations on human monocyte chemotaxis,
the migratory response of SMCs toward SN was observed to be similar for
both the human and rat SN preparations (data not shown). To further
define the neuropeptide's structural requirements for mediating SMC
chemotaxis, assays were conducted with the carboxyl-terminal fragment
of SN. The carboxyl-terminal fragment, too, significantly stimulated
SMC migration in a range from 10-12 to 10-6
M, and the response showed the same maximum at 10-10 M
(Fig 2). The results suggest that the
carboxyl-terminus of the peptide is involved in the migratory response.
These findings are consistent with previous chemotactic studies
on human monocytes and human skin fibroblasts.27 28 In
comparison with PDGF AB (10 ng/mL), SN and its carboxyl-terminal
fragment stimulated SMC chemotaxis at maximal effective concentrations
to a higher extent (145% of the observed PDGF effect). In contrast,
two other secretogranin II fragments, bEL and bLF, exhibited no effect
on SMC migration (Fig 2). To further investigate whether the observed
stimulation of SMC migration is specific, we tested chemotaxis toward
SN in the presence of antibodies. The specific rabbit anti-SN serum
(dilution, 1:1000) completely abolished the SN (10-10 M)
effect, whereas the antiserum itself did not affect SMC migration (Fig 3). These data indicate that the
neuropeptide SN serves as a chemotactic agent for SMC in vitro.
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Secretoneurin Stimulates SMC DNA Synthesis and Cell Growth
Incubation of SMCs with DMEM containing 0.5% FCS alone (control
conditions) had no significant effect on cell growth and DNA synthesis
for up to 8 days of postinoculation as measured by MTT assay and 5-BrdU
incorporation. SMCs were made quiescent by normal serum starvation over
3 days. The growth experiments were performed in the presence of 0.5%
FCS to minimize possible interactions of SN with serum-associated
mitogens. In initial experiments, we compared the effect of different
concentrations of FCS (0%, 0.2%, 0.5%, and 2.5%) on cell growth,
survival, and DNA synthesis of quiescent SMC cultures in the absence of
known mitogens. In these preliminary studies, medium supplemented with
0.5% FCS allowed optimal growth stimulation of synchronized cells if
well defined mitogens such as epidermal growth factor were used (data
not shown).
SN, when added to medium complemented with 0.5% FCS, switched SMCs
from the quiescent state and significantly enhanced DNA synthesis after
48 hours of incubation (Fig 4).
Stimulation of DNA synthesis was observed at a concentration range from
10-12 to 10-6 M of SN with a maximal response
at 10-8 M. A bell-shaped curve was obtained also with the
carboxyl-terminal fragment of SN, with a maximum at a concentration of
10-8 M. Bell-shaped, dose-response curves are typically
seen when receptor down-regulation occurs at high agonist
concentrations. Our deactivation of secretoneurin at high
concentrations suggests involvement of receptor-dependent mechanisms.
The observed increase in DNA synthesis was comparable with the effect
of PDGF AB (10 ng/mL) on SMC DNA synthesis. PDGF AB, too, caused
a significant enhancement of DNA synthesis in a concentration-dependent
manner, with a maximum at 10 ng/mL. To further investigate
whether the observed effect is specific for SN, we coincubated SMC
monolayers with SN and the specific rabbit anti-SN serum (Fig 5). In the presence of the antibodies at
a final dilution of 1:1000, SN failed to stimulate significant DNA
synthesis compared with control conditions. Controls included a
rabbit-irrelevant nonspecific antiserum as well as incubation with the
specific antiserum alone. The specific antiserum itself had no effect
on DNA synthesis. Furthermore, there was no significant alteration when
we compared SN with the effect of SN coincubated with the nonspecific
antiserum (Mann-Whitney U test, P>0.05). In
contrast to the above effects of SN on SMC DNA synthesis, two other
secretogranin II fragments (bEL-17, bLF-19) exhibited no stimulatory
effect (Fig 4).
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Furthermore, SN increased optical density in the MTT assay in a
time-dependent manner, with a significant stimulation of cell growth
after 3 days of incubation (Fig 6).
Growth stimulation was observed at a concentration range similar to the
DNA synthesis experiments (from 10-12 to 10-6
M SN). The increase in optical density of SN-induced proliferation in
MTT experiments was similar to the one in the identical PDGF
experiments. The maximum response toward PDGF AB was seen at 10
ng/mL. Two other secretogranin II fragments, bEL-17 and bLF-19,
and a trypsinized SN peptide exhibited no significant effect on SMC
growth (the Table), whereas the 15-amino
acid carboxyl-terminal fragment increased optical densities as measured
colorimetrically (the Table).
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Discussion |
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Recent advances in the understanding of the biology of arteriosclerosis and restenosis indicate that these events are predominantly caused by a multifactorial stimulation of SMC proliferation and migration, even if it was shown that vascular SMCs do not represent a homogenous population and despite the lack of direct evidence for cell multiplication of SMCs in human arteriosclerotic lesions up to the present day.38 39 Although many growth regulatory molecules and cytokines may be formed within the atherosclerotic lesions, a few play dominant roles in this process: both chains of PDGF;40 41 42 43 two forms of colony stimulating factor (CSF), M-CSF and GM-CSF;44 45 basic fibroblast growth factor;46 47 48 49 50 51 insulin-like growth factor-I;52 transforming growth factor-ß (TGF-ß53 54 55 56 ); the interleukin-1;57 58 59 and tumor necrosis factor-
.60 61 62 In contrast to these
proliferative stimuli, TGF-ß is perhaps the most potent inducer of
the synthesis of connective tissue matrix macromolecules (e.g.,
collagens, proteoglycans, and elastic fiber proteins) but inhibits SMC
growth and migration.55 61 62 63 Furthermore, adult human
aortic SMCs in culture produce TGF-ß as an autocrine
inhibitory loop,64 and also heparins act via
releasing TGF-ß.65 As atherosclerotic plaques contain a large number of SMCs, it has been suggested that migration of vascular SMCs from the media to the intima is a precondition for subsequent SMC proliferation and intimal thicking. Several growth factors that are both mitogens and chemoattractants are involved in this process.46 The first aim of the present study, therefore, was to investigate the role of SN in SMC migration. In comparison with PDGF-AB, which caused high levels of migration, SN elicited a significantly more potent migratory response, with a maximum at 10-10 M. Investigations demonstrated SN-directed migration as being abolished by specific SN antibodies. Additionally, the migratory response of SMC toward two other secretogranin II fragments, namely bLF-19 and bEL-17, was tested. As observed in previous experiments with human monocytes27 and human skin fibroblasts,28 both proteins showed no effect on SMC migration. The maximal chemotactic response of SMC toward SN was seen at doses similar to the optimal concentrations, causing migration of human monocytes27 and human skin fibroblasts.28 These data suggest the possible involvement of this newly discovered neuropeptide in the regulation of SMC migration.
Despite the lack of direct evidence for cell multiplication, proliferation of SMCs in human arteriosclerotic lesions has been assumed to play an important role in the initiation and progression of human atherosclerosis. Growth factors may accelerate proliferation of vascular SMCs in an autocrine and/or paracrine manner. The functional changes of SMCs correlate with the modulation of the SMC phenotype from a quiescent, contractile state to a synthetic, proliferative state. As SN may be present in sensory nerve endings within the vascular wall and can be determined in human plasma, a further intention of this study was to explore whether the recently discovered neuropeptide SN is able to affect SMC proliferation in vitro. Results indicate that SN is able to stimulate both an increase in cell number and in DNA synthesis in vitro. SN induced SMC proliferation under serum-deprived conditions. Increasing optical density was observed with increasing SN concentrations up to 10-8 M, whereas a further increase showed less effect on both cell proliferation as measured by MTT assay and DNA synthesis measured by BrdU incorporation. In both experiments, we found a maximal response of SMC to SN occurring at a concentration of 10-8 M. The peak response toward SN in the DNA experiments occurred after 48 hours, suggesting a delayed entry of SMC into S-phase, as described for thrombin and substance P.66 Furthermore, the mitogenic potency was comparable with the one toward PDGF-AB. We were able to demonstrate the specificity of the observed neuropeptide action by blocking SN-induced proliferation and DNA synthesis by specific antibodies. As both human and rat SN were active on SMCs, no species specificity seems to exist. Specificity of SN effects is further supported by the lack of activity of secretogranin II fragments other than SN. As SMC migration and proliferation determine whether a lesion progresses from a fatty streak to a fibrous plaque that can occlude an artery, SN may play a critical role in the pathogenesis of atherosclerotic plaques.
In conclusion, the present study demonstrates that SMCs are a potential target for SN. SN is a mitogen and chemoattractant for aortic smooth muscle cells in vitro with a maximum proliferative response at 10-8 M and 10-10 M for chemotaxis. If SN is released in sufficient amounts within the vessel wall, these observations may be of relevance in the pathogenesis of atherosclerosis. The role of monocytes in atherogenesis has been recently suggested, including transformation into macrophages, lipid uptake, secretion of mitogenic factors, and generation of toxic products. Interestingly enough, we recently reported that SN can affect monocyte and fibroblast functions.27 28 We hypothesize that under circumstances of vascular injury, SN stimulates the directional migration of medial SMCs into the intima and augments their proliferation after having been released from sensory nerve endings.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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Supported by the Austrian Science Fund FWF no. 9977 and by the Austrian National Bank. The authors thank R. Fischer-Colbrie, M.D., and Prof. H. Winkler, M.D., from the Department of Pharmacology, Faculty of Medicine, University of Innsbruck, for fruitful discussions and for kindly providing secretoneurin reagents.
Received June 27, 1996; accepted February 6, 1997.
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