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Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:2029-2035

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:2029-2035.)
© 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

Christian M. Kähler; Peter Schratzberger; ; Christian J. Wiedermann

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.


*    Abstract
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 *Abstract
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 down arrowMaterials
 down arrowResults
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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


*    Introduction
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 up arrowAbstract
 *Introduction
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 down arrowResults
 down arrowDiscussion
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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.


*    Materials
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 up arrowAbstract
 up arrowIntroduction
 *Materials
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
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.


*    Results
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 up arrowIntroduction
 up arrowMaterials
 *Results
down arrowDiscussion
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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 1Down), 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 2Down). 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 2Down). 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 3Down). These data indicate that the neuropeptide SN serves as a chemotactic agent for SMC in vitro.



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  		Figure 1. Dose-response curve of secretoneurin on SMC migration. SMC migration was examined in the Transwell cell culture chamber as described under "Materials and Methods." The number of cells that had migrated to the lower surface of the filters was determined colorimetrically. The concentration of PDGF AB was 10 ng/mL, and the incubation time 4 hours. Values represent the mean±SEM, (n=5). The mean optical density of the vehicle control was 0.054±0.008. *P<0.05; **P<0.01 versus basal level.



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  		Figure 2. Dose-response curve of the C-terminal fragment of secretoneurin and two other secretogranin II cleavage products (LF-19, EL-17) on SMC migration. SMC migration was examined in the Transwell cell culture chamber as described under "Materials and Methods." The number of cells that had migrated to the lower surface of the filters was determined colorimetrically. The concentration of PDGF AB was 10 ng/mL, and the incubation time 4 hours. Each point represents the mean±SEM, (n=5). The mean optical density of the vehicle control was 0.051±0.004. *P<0.05; **P<0.01 versus basal level.



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  		Figure 3. Effect of antisecretoneurin antibody (1:1000) on secretoneurin-induced (10-8 M) SMC migration. SMC migration was examined in the Transwell cell culture chamber as described under "Materials and Methods." The number of cells that had migrated to the lower surface of the filters was determined colorimetrically. The concentration of PDGF AB was 10 ng/mL, and the incubation time 4 hours. Values represent the mean±SEM, (n=5). The mean optical density of the vehicle control was 0.056±0.008. *P<0.05 versus SN alone.

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 4Down). 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 5Down). 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 4Down).



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  		Figure 4. Dose-response curve of secretoneurin, its carboxyl-terminal fragment, and two other secretogranin II fragments (bEL-17 and bLF-19) on SMC DNA synthesis. SMCs made quiescent in 0.2% serum were switched to fresh 0.5% serum when secretoneurin (n=12), its carboxyl-terminal fragment (n=4), bEL-17 (n=4), bLF-19 (n=4), PDGF AB (10 ng/mL, n=12) were added. DNA measurement was carried out after further 48 hours of incubation. DNA synthesis was measured colorimetrically. Values represent the mean±SEM, minus optical density of the medium control. The mean optical density of the medium was 0.401±0.044. *P<0.05; **P<0.01; ***P<0.001 versus medium control.



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  		Figure 5. Effect of antisecretoneurin antibody (1:1000) on secretoneurin-induced (10-8 M) SMC DNA synthesis. SMCs made quiescent in 0.2% serum were switched to fresh 0.5% serum when secretoneurin, PDGF AB (10 ng/mL), and/or anti-SN antibody were added. DNA measurement was carried out after further 48 hours of incubation and measured colorimetrically. Values represent the mean±SEM, (n=8). *P<0.05 versus vehicle control.

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 6Down). 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 TableDown), whereas the 15-amino acid carboxyl-terminal fragment increased optical densities as measured colorimetrically (the TableDown).



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  		Figure 6. Dose-response curve of secretoneurin on SMC proliferation. SMCs made quiescent in 0.2% serum were switched to fresh 0.5% serum when secretoneurin or PDGF AB (10 ng/mL) were added. The MTT assay was carried out after further 72 hours of incubation. Values represent the mean±SEM (n=12) minus optical density of the vehicle control. The mean optical density of the vehicle control was 0.456±0.013. *P<0.05; **P<0.01; ***P<0.001 versus vehicle control.


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  		Table 1.


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials
 up arrowResults
 *Discussion
down arrowReferences
 
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-{alpha}.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.


*    Selected Abbreviations and Acronyms
 
DMEM = Dulbecco's modified Eagle medium
FCS = fetal calf serum
PDGF = platelet-derived growth factor
SMC = smooth muscle cell
SN = secretoneurin
TGF = transforming growth factor


*    Acknowledgments
 
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.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials
 up arrowResults
 up arrowDiscussion
 *References
 

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