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2 and endothelin
Departments of Pharmacology, University of Texas Health Science Center, San Antonio, Texas 78284; and University of Vermont, Burlington, Vermont 05405
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Sympathetic nerves are purported to stimulate blood
vessel growth. The mechanism(s) underlying this stimulation has not
been determined. With use of an in vitro coculture model, the present study tests the hypothesis that sympathetic neurons stimulate the
growth of vascular smooth muscle (VSM) and evaluates potential mechanisms mediating this stimulation. Sympathetic neurons isolated from superior cervical ganglia (SCG) stimulated the growth of VSM.
Growth of VSM in the presence of SCG (856 ± 81%) was significantly greater than that in the absence of SCG (626 ± 66%,
P < 0.05). SCG did not stimulate VSM
growth in transwell cocultures. An antibody that neutralized the
activity of transforming growth factor-
2 (TGF-2) inhibited SCG
stimulation of VSM growth in coculture. SCG stimulation of VSM growth
was also inhibited by an endothelin A receptor antagonist. These data
suggest novel mechanisms for sympathetic modulation of vascular growth
that may play a role in the physiological and/or pathological growth of
the vasculature.
blood vessels; sympathetic nervous system; growth factor; vascular
smooth muscle; transforming growth factor-2
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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DURING DEVELOPMENT, blood vessels must clearly grow to match the vascular supply to tissue metabolic demands. In the healthy adult animal, there is little vascular growth but many cardiovascular diseases, including hypertension (17) and atherosclerosis (12, 22), are characterized by aberrant vascular growth that may compromise cardiovascular function. The mechanisms that regulate physiological and/or pathological vascular growth are not well understood.
In vivo studies indicate that sympathetic innervation promotes vascular growth (4, 12, 17, 23, 31). The mechanism underlying this action has not been identified. In vitro studies suggest multiple potential mechanisms that may mediate sympathetic stimulation of vascular growth. Activation of sympathetic neurons causes the release of neurotransmitters and cotransmitters that have been shown to stimulate the growth of vascular smooth muscle (VSM) and endothelial cells (EC) (5, 23, 28, 32). In addition, many neurons, including sympathetic neurons, produce growth factors, the release of which is not dependent on neuronal activity (6).
In the present study, a sympathetic neuron/VSM coculture model is used to determine if and how postganglionic sympathetic neurons stimulate the growth of VSM.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Materials.
Dulbecco's modified essential medium (DMEM), penicillin-streptomycin,
and glutamine were purchased from GIBCO Life Technologies. Fetal bovine
serum (FBS) was purchased from Summit Biotechnology. Rat tail collagen
and nerve growth factor (NGF) were purchased from Collaborative
Biomedical Products. Collagenase (type 2), hyaluronidase, and trypsin
were purchased from Worthington Biochemical. Mitomycin C and smooth
muscle 
-actin antibody were purchased from Sigma. Rhodamine-labeled
secondary antibody was purchased from Boehringer Mannheim Chemicals.
Tyrosine hydroxylase (TH) primary antibody and fluorescein-labeled
secondary antibody were purchased from Chemicon International.
Vascular cell culture.
EC isolated from adult (>90 days) male Sprague-Dawley rats were a
generous gift from Dr. Paula Grammas (University of Oklahoma). These
cells exhibited the distinct cobblestone morphology characteristic of
EC and they took up acetylated low-density lipoprotein
(27). EC were used from passages
11-20. VSM was isolated from explants of adult
male Sprague-Dawley rat aortas (21). These cells exhibited characteristic "hill and valley" growth patterns and
immunohistochemical labeling with a monoclonal antibody for smooth
muscle-specific -actin. VSM was used from passages
3-10. Vascular cells were grown in low glucose
DMEM supplemented with 10% FBS, 1 mM glutamine, 100 units penicillin,
and 100 units streptomycin. Cells were maintained at 37°C in a
humidified 5% CO2 environment.
Superior cervical ganglion cultures.
Rat pups (3-4 days of age, male and female, Sprague-Dawley,
Harlan) were anesthetized with metofane and euthanized by removing their hearts. Sympathetic superior cervical ganglia (SCG) were collected and enzymatically dissociated for 20 min at 37°C in a
collagenase-hyaluronidase solution (10 mg/ml bovine serum albumin, 4 mg/ml collagenase, 1 mg/ml hyaluronidase) and then for 3 min in trypsin
(3 mg/ml). Dissociated cells were applied to collagen-coated dishes.
Neuronal cultures (SCG) were grown in DMEM supplemented with 10% FBS,
50 ng/ml NGF, penicillin-streptomycin, and glutamine. Nonneuronal
cultures [SCG(
)] were grown in DMEM supplemented with 10% FBS, penicillin-streptomycin, and glutamine without NGF. NGF
is absolutely required for the survival of sympathetic neurons, and
thus these cultures contained nonneuronal SCG cells but not neurons.
SCG and SCG() cultures were maintained at 37°C in a humidified 5% CO2 environment.
SCG/vascular and SCG()/VSM cocultures.
One day after plating, SCG and SCG() cultures were treated with
an antimitotic agent (mitomycin C, 10 µg/ml for 1 h) to prevent the
growth of nonneuronal cells. After removal of the mitomycin C, VSM or
EC was added to SCG cultures and VSM was added to SCG() cultures
and allowed to attach overnight.
Transwell SCG/VSM cocultures. SCG were plated in a 24-well tissue culture dish. One day after plating, the SCG were growth arrested (10 µg/ml mitomycin C for 60 min). After removal of the growth-arresting agent, VSM (1 × 104 cells/well) was plated on a transwell insert (0.40 µm pores), which sits 1 mm above the well containing the SCG. Thus the SCG and VSM are cultured in the same medium and can reciprocally exchange soluble effectors, but they are not in close proximity and cannot make physical contact. As a control, VSM was also grown on the transwell insert but in the absence of SCG.
Immunohistochemistry.
Four-day SCG/VSM cocultures were rinsed with 0.1 M phosphate-buffered
saline (PBS, 19 mM sodium phosphate monobasic, 81 mM sodium phosphate
dibasic, 0.05 sodium chloride, pH 7.4) and fixed for 2 h in 4%
paraformaldehyde in 0.1 M PBS. The cells were then permeabilized (1 h
in 0.1 M PBS, 0.2% Triton X-100, 0.9% hydrogen peroxide) and blocked
(30 min in 5% normal goat serum). To label sympathetic neurons, cells
were incubated with TH (rate-limiting enzyme in catecholamine
synthesis) primary antibody (rabbit, 1:4,000) overnight at room
temperature, washed with 0.1 M PBS, and incubated with a
fluorescein-labeled secondary antibody (donkey anti-rabbit IgG
fluorescein, 1:200) overnight at 4°C. To label VSM, cocultures were
then incubated with smooth muscle -actin primary antibody (mouse,
1:400) for 1 h at 37°C, washed, and then incubated with a
rhodamine-labeled secondary antibody (goat anti-mouse IgG rhodamine, 1:10) for 1 h at 37°C. Immunofluorescence was visualized with a
Nikon diaphot microscope with appropriate fluorescent filters.
Fluorescence imaging of intracellular calcium. For these experiments the SCG, VSM, and SCG/VSM cultures were placed in HEPES-buffered saline, pH 7.4, and loaded with 1 µM of the membrane permeant Ca2+ indicator dye, fura 2 (the acetoxymethyl ester form) for 30 min at room temperature. Excess dye was removed by washing the cells twice. The cells were then incubated for 15 min at room temperature to allow cytosolic esterases to cleave the ester rendering the dye impermeant. Calcium in the cells was visualized with a Nikon Diaphot microscope equipped for epifluorescence coupled to a Hamamatsu silicon-intensified target camera. The images were recorded and analyzed with an IMAGE-1/FL quantitative fluorescent measuring program (Universal Imaging). Fluorescent ratio images of 510 nm emissions resulting from 340 and 380 nm excitation were acquired. This fluorescence ratio was measured as an index of free cytosolic calcium.
Proliferation assays.
For cell growth assays, all cells were plated in 24-well
collagen-coated dishes. Cell growth was assayed in VSM and EC cultures and in SCG/VSM, SCG/EC, and SCG()/VSM cocultures. Cell growth was assessed as increases in cell number. Cell numbers were counted electronically (Coulter Electronics). For growth-arrested cells, the
effectiveness of growth arrest was verified by counting cell number
before and 5 days after growth arrest. For all cultures and cocultures,
approximately 1 × 104
cells/well were plated. For all cultures and cocultures, the day after
all cells were plated, three wells were counted to determine the
starting cell number (day 0 cell
count). The remaining wells of cells were then placed in DMEM
supplemented with 5% FBS, penicillin-streptomycin, glutamine, and 50 ng/ml NGF and allowed to grow for 4 days (5% FBS submaximally
stimulated the growth of VSM and EC and thus inhibitory and/or
stimulatory effects could be assessed). The cells were then counted
(day 4 cell count), and the percent
increases in VSM or EC number were determined. {For the VSM or
EC cultures, the percent increase in cell number = [(day 4 VSM or EC count day 0 VSM or EC
count)/day 0 VSM or EC count] × 100. For the cocultures, the percent increase in VSM or EC
number = [(day 4 coculture count day 0 coculture
count)/day 0 VSM or EC count] × 100}. In the cocultures, because the SCG or SCG()
cells were growth arrested, any increase in cell number is attributable
to increases in VSM or EC number. Sympathetic stimulation of vascular cell growth was calculated as percentage increases in cell number in
the presence of SCG minus percent increases in cell number in the
absence of SCG.
Northern analysis.
RNA was isolated as described by Chirgwin et al. (7). Briefly, cells
that had been grown in culture for 4 days were lysed with 4 M guanidium
isothiocyanate centrifuged through a gradient of cesium chloride.
Pelleted RNA was purified by chloroform-butanol extraction and ethanol
precipitation, separated by electrophoresis through an agarose
(1.2%)-formaldehyde gel, and transferred to nitrocellulose. The RNA
was immobilized on the nitrocellulose by baking for 2 h at 80°C.
After prehybridization, RNA was labeled by hybridization to
radioactively labeled cDNAs encoding for transforming growth
factor-1 (TGF-1) [obtained from American Type Culture Collection (ATCC); 3], TGF-2 (obtained from ATCC; 18), and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (13). After
hybridization, nitrocellulose membranes were washed under stringent
conditions and exposed to X-ray film.
Statistical analysis. All data are expressed as means ± SE. All growth experiments were performed in triplicate; the triplicate data were averaged for each number. Unpaired t-tests were used to determine statistically significant differences (P < 0.05).
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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SCG/VSM cocultures.
The goal of the present studies was to determine if and how sympathetic
neurons modulate the growth of VSM. An in vitro coculture model was
used to achieve this goal. These cultures contained postganglionic
sympathetic neurons and a small percentage (10-20%) of
nonneuronal cells. The SCG cells were growth arrested to prevent the
growth of nonneuronal cells, and then VSM was added to the cultures.
The initial ratio of neurons to VSM in the cocultures was approximately
1:1. Figure 1 shows a representative
coculture immunohistochemically labeled with TH (green fluorescence),
the rate-limiting enzyme in catecholamine synthesis and thus a marker for postganglionic sympathetic neurons, and smooth muscle -actin (red fluorescence), a marker for VSM. Note that in Fig. 1 no VSM exhibited green fluorescence and no sympathetic neurons exhibited red
fluorescence, indicating that the immunohistochemical labeling with
both TH and smooth muscle -actin was specific.
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4 M NE. Fluorescent
ratios before the addition of NE were 0.4 ± 0.006 (means ± SE).
The addition of NE increased the fluorescent ratio to 2.09 ± 0.19 (peak ratio). This indicates that the sympathetic neurotransmitter NE
could act on these cells. The addition of ACh
(104 M) did not increase
intracellular calcium in any VSM cultured in the absence of SCG neurons
(data not shown), suggesting that ACh does not directly act on VSM to
regulate intracellular calcium. The addition of ACh to SCG grown in the
absence and presence of VSM increased calcium in SCG neurons (Fig.
2, solid line), suggesting that ACh would
promote the release of neurotransmitter from these neurons. Fluorescent
ratios measured in five neurons were 0.43 ± 0.04 before and 1.77 ± 0.24 after the addition of ACh. In three independent
cocultures, ACh increased intracellular calcium in VSM (Fig. 2, dotted
line), suggesting that ACh can indirectly modulate intracellular
calcium in VSM by activating the SCG neurons. For these VSM
(n = 3), the fluorescent
ratios were 0.45 ± 0.07 before and 1.25 ± 0.18 after the
addition of ACh. Thus the morphological data in Fig. 1 and the
functional data in Fig. 2 suggest that in the coculture model used in
the present study, postganglionic sympathetic neurons make functional
synapses with VSM.
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VSM growth is stimulated in SCG/VSM cocultures.
VSM growth (%increase in VSM number) in the presence of sympathetic
neurons (presence of SCG, 856 ± 81%) was greater than that in the
absence of sympathetic neurons (absence of SCG, 626 ± 66%;
P < 0.05; Fig.
3A),
suggesting that sympathetic neurons stimulate the growth of VSM in
SCG/VSM cocultures. The difference between growth in the presence of
SCG and that in the absence of SCG (231 ± 54%) is the stimulation
attributable to the SCG neurons. Sympathetic neurons did not stimulate
the growth of EC in SCG/EC cocultures (Fig.
3B). Growth of EC in the presence of SCG (943 ± 159% increase) was not significantly different from that in the absence of SCG (849 ± 70% increase;
P > 0.05).
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)/VSM cocultures. The SCG() cultures contain nonneuronal superior
cervical ganglion cells but no postganglionic sympathetic neurons. VSM growth was not stimulated in SCG()/VSM cocultures (Fig.
3C). In fact, VSM growth in the
presence of nonneuronal superior cervical ganglion cells
[presence of SCG(), 95 ± 16% increase] was less than that in the absence of these cells [absence of SCG(),
267 ± 60% increase].
The effects of SCG on VSM growth were also assessed in transwell
cocultures. In these cocultures SCG and VSM are cultured in the same
medium and thus can reciprocally exchange stable soluble mediators, but
the cells are separated by 1 mm, which prevents physical contact and/or
close proximity. In this coculture system, SCG did not stimulate VSM
growth (Fig. 4, solid bar). The lack of
stimulation in transwell cocultures is in contrast to the stimulation that was observed in parallel SCG/VSM cocultures that allowed physical
contact and/or close proximity (Fig. 4, open bar).
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TGF-2 and endothelin are required for SCG
stimulation of VSM growth in SCG/VSM cocultures.
Figure 3 indicates that SCG neurons stimulate the growth of VSM in
cocultures in which the two cell types are in contact or close
proximity. The data in Fig. 4 indicate that close proximity or cell
contact is required for this stimulation. TGF- is produced by
neurons (11, 26) and VSM (18, 20), is activated by heterotypic cell
interactions that require close proximity or cell contact (2), and
stimulates VSM growth (25, 30). Does TGF- mediate SCG stimulation of
VSM growth in SCG/VSM cocultures? Neutralization of the activity of
TGF-2 with an antibody (goat) inhibited SCG stimulation of VSM
growth in SCG/VSM cocultures (Fig. 5).
Growth of VSM in the absence of SCG was not affected by the TGF-2
antibody (425 ± 32% increase in the absence vs. 406 ± 34%
increase in the presence of the antibody;
P > 0.05), but growth of VSM in the
presence of SCG was inhibited (589 ± 68% increase in the absence
of antibody vs. 445 ± 27% increase in the presence of antibody;
P < 0.05). In the presence of the TGF-2 neutralizing antibody, VSM growth in the presence of SCG (445 ± 27%) was not significantly greater than that in the absence of
SCG (406 ± 34%). A nonimmune goat IgG (data not shown) and an
antibody that neutralized the activity of TGF-1 did not alter VSM
growth in the presence or absence of SCG, and thus these antibodies did
not modulate SCG stimulation of VSM growth (Fig. 5).
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2 is required for sympathetic
stimulation of VSM growth in SCG/VSM cocultures. Thus the data in Fig.
5 also suggest that TGF-2 is produced in SCG/VSM cocultures.
Northern analysis of TGF- expression in SCG, VSM, and SCG/VSM
cultures supports this suggestion. TGF-2 mRNAs (4.0 and 5.0 kb) were
expressed in SCG, VSM, and SCG/VSM cultures (Fig. 6A).
Under equivalent experimental conditions, expression of TGF-1 mRNA
(2.5 kb) was undetectable, although this mRNA was easily detected in
cultures of bovine EC (Fig.
6B). GAPDH mRNA was
detectable in all samples.
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2 can stimulate the growth of VSM by increasing the production
of VSM mitogens (14, 25). Endothelin is a VSM mitogen (1, 29) that is
produced by postganglionic sympathetic neurons (9) and VSM (15), and
TGF- increases VSM production of endothelin-1 (15). Thus the
sympathetic stimulation of VSM growth observed in SCG/VSM cocultures
could be mediated by TGF-2 and endothelin-1. To test this
hypothesis, VSM growth was assessed in the presence and absence of
endothelin receptor A antagonists BQ-123 and BQ-610. Similar results
were obtained with these two agents, and thus the data were combined.
The addition of 1 µM BQ-123 (or BQ-610) inhibited sympathetic
stimulation of VSM growth in SCG/VSM cocultures (Fig.
7).
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2
and that the effects of TGF-2 are mediated by endothelin. If this is
the case, then TGF-2 should stimulate the growth of VSM in the
absence of SCG, and endothelin antagonism should prevent this
stimulation. The effects of TGF-2 and BQ-123 on VSM growth in the
absence of SCG are shown in Fig. 8. In the
absence of BQ-123, 10 ng/ml TGF-2 stimulated VSM growth (44.6 ± 24.9% increase in cell number, open bar). In the presence of 10 µM
BQ-123, TGF-2 no longer stimulated and in fact inhibited VSM growth
(41.0 ± 22.4% decrease in cell number, solid bar).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The majority of blood vessels in animals and humans are innervated by the sympathetic nervous system. It is well known that sympathetic innervation modulates the contractile activity of the vasculature and is thus an important determinant of blood pressure (17). Sympathetic innervation also has been reported to modulate the growth of blood vessels (4, 12, 17, 24, 31), but much less is known about this action or the mechanism of this action. The data presented demonstrate that postganglionic sympathetic neurons stimulate the growth of VSM in an in vitro coculture system and identify novel mechanisms whereby sympathetic neurons may act on VSM.
An in vitro coculture model was used to study sympathetic stimulation of VSM growth. The cocultures contained postganglionic sympathetic neurons and a small percentage of nonneuronal cells isolated from SCG of neonatal rats and vascular cells from adult rat aortas. The sympathetic and vascular cells were cultured in the same dish in close proximity and/or direct contact. In these cultures, the sympathetic neurons could modulate vascular cell growth by releasing growth factors from their cell bodies and/or nerve terminals.
Figure 1 suggests that in the cocultures used in the present studies, processes from the neurons make contact with the VSM. Do they actually make synapses? If the neurons were making functional synapses with the smooth muscle cells, selective activation of the neurons should cause the release of neurotransmitter, which would then act on the smooth muscle. Fluorescence imaging of intracellular calcium was used to test this hypothesis. ACh was used to selectively activate the neurons. ACh increased intracellular calcium in SCG neurons cultured in the presence and absence of VSM, suggesting that addition of ACh to the cultures would promote the release of NE from the neurons. ACh did not alter intracellular calcium in VSM cultured in the absence of SCG, indicating that ACh did not directly modulate intracellular calcium in these cells. NE increased intracellular calcium in VSM cultured in the absence of SCG, indicating that the VSM could respond to this neurotransmitter if it was released from SCG neurons in the SCG/VSM cocultures. Addition of ACh to SCG/VSM cocultures increased intracellular calcium in VSM as well as SCG neurons (Fig. 2), suggesting that functional synapses were present. The frequency of SCG/VSM synapse formation was not assessed in the present study but is currently under investigation.
VSM growth was stimulated in SCG/VSM cocultures (Fig. 3A). SCG/VSM coculture produced a 230 ± 54% increase in VSM number. This increase is comparable to that induced by maximally active concentrations of other VSM mitogens (10, 29). EC growth was not stimulated in SCG/EC cocultures (Fig. 3B), indicating that SCG produce a factor or factors that stimulate the growth of VSM but not EC.
SCG cultures used in the present study contained postganglionic
sympathetic neurons as well as a small number of nonneuronal cells. SCG
stimulation of VSM growth could be mediated by either or both of these
cell types. To determine whether nonneuronal SCG could stimulate VSM
growth, VSM was cocultured with SCG cultures that had been grown in the
absence of NGF [SCG()]. These SCG() cultures
contained nonneuronal cells but no neurons. VSM growth was not
stimulated in SCG()/VSM cocultures (Fig.
3C). In fact, VSM growth was
inhibited, indicating that SCG stimulation of growth in SCG/VSM
cocultures requires postganglionic sympathetic neurons and that the
presence of the neurons overrides any inhibitory effects associated
with the nonneuronal cells.
VSM growth was not stimulated in SCG/VSM transwell cocultures (Fig. 4). In these cocultures, the cells are cultured in the same medium and thus can reciprocally exchange stable soluble mediators, but the VSM and the SCG are separated by 1 mm and thus are not in close proximity or contact. Thus SCG stimulation of VSM growth in coculture is dependent on proximity or contact between SCG and VSM.
TGF- has been reported to stimulate VSM growth (25, 30), is produced
by vascular cells (16, 18, 20) and many neurons (11, 26), and is
activated when heterotypic cells are in close proximity or contact (2).
Thus TGF- was a likely mediator of the SCG stimulation of VSM growth
observed in the present studies. When SCG and VSM were cocultured in
the presence of an antibody that neutralized the activity of TGF-2,
SCG no longer stimulated the growth of VSM (Fig. 5). SCG did stimulate
VSM growth when cocultures were grown in the presence of a nonimmune
goat IgG (data not shown), indicating that the effect of the TGF-2
antibody was attributable to the neutralization of the activity of
TGF-2. The ability of SCG to stimulate the growth of VSM in
coculture was also unaffected by antibodies that neutralized the
activity of TGF-1 (Fig. 5).
The present studies using neutralizing antibodies suggest that active
TGF-2 but not TGF-1 is produced in VSM, SCG, and SCG/VSM cultures. Consistent with previous observations (18, 20), mRNA encoding
for TGF-2 was detected in VSM (Fig.
6A). TGF-2 mRNA was also detected
in SCG cultures and in SCG/VSM cocultures (Fig.
6A). This isoform of TGF- is
known to be expressed by many other neurons (11, 26), but this is the
first report that it is expressed by postganglionic sympathetic
neurons. The amount of TGF-1 mRNA in VSM, SCG, and SCG/VSM cultures
was considerably less than that of TGF-2 mRNA (Fig.
6B). In the present studies, Northern analysis was performed on cultures in which the VSM was confluent. These cultures would be comparable to the high density VSM
cultures used by Hamet et al. (16), in which TGF-1 mRNA expression
was also very low. The preferential expression of TGF-2 and not
TGF-1 by sympathetic neurons is consistent with previous reports of
TGF- isoform expression in other neurons (11, 26).
The data in Fig. 7 indicate that endothelin A receptor antagonism also
inhibits stimulation of VSM growth in SCG/VSM cocultures. Endothelin,
acting at endothelin A receptors, is a mitogen for VSM (1, 29) that is
produced by postganglionic sympathetic neurons (9) and rat VSM (15).
TGF- is known to increase VSM endothelin-1 mRNA and peptide
expression (15). Thus the data in Figs. 5-7 support the hypothesis
that TGF-2 is produced in SCG/VSM cocultures and stimulates the
production of endothelin, which stimulates the growth of VSM. Further
support for this hypothesis is provided by the data shown in Fig. 8.
VSM growth in the absence of SCG was slightly stimulated by TGF-2.
This stimulation was inhibited by BQ-123, an endothelin A receptor
antagonist. In fact, in the presence of BQ-123, TGF-2 inhibited
rather than stimulated VSM growth.
In vivo studies clearly indicate that sympathetic innervation modulates
the physiological growth of VSM that occurs during development (4, 31)
and the pathological growth that occurs in hypertension (17) and in
response to vascular injury (12). The goal of the present studies was
to determine the mechanism whereby sympathetic innervation modulates
VSM growth. An in vitro coculture model was used to achieve this goal.
Several lines of evidence suggest that this model is appropriate for
studying sympathetic regulation of VSM growth. SCG neurons grown in
culture are biochemically and electrophysiologically similar to those
in vivo (8). The VSM used in the present studies, which was isolated
from adult rat aortas, have growth properties similar to those of VSM
in developing arteries and in injured arteries (22). Two differences between in vivo and the in vitro model should be noted. In vivo, only
postganglionic sympathetic nerve terminals (not cell bodies) would be
in close proximity to VSM; in vitro, VSM has access to both cell bodies
and nerve terminals. Also, in the presently used in vitro model, the
neurons were not stimulated; in vivo, the neurons would be
intermittently stimulated. Studies are in progress to extend the
current in vitro observations, and determine the role of TGF-2 and
endothelin in sympathetic regulation of VSM growth in vivo.
In summary, the present studies demonstrate that postganglionic
sympathetic neurons stimulate the growth of VSM in cocultures that
allow close proximity or direct contact between the neurons and VSM.
Data are also presented demonstrating that the VSM, SCG, and SCG/VSM
cultures express mRNA encoding for TGF-2, and that if the activity
of TGF-2 is inhibited, the SCG no longer stimulate VSM growth.
Finally, inhibition of endothelin binding to endothelin A receptors is
also shown to prevent SCG stimulation of VSM growth in SCG/VSM
cocultures, suggesting that endothelin and TGF-2 are required for
SCG stimulation of VSM growth.
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ACKNOWLEDGEMENTS |
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I acknowledge the expert technical assistance of Zhuan Chen and thank Dr. Mark Nelson for the use of the calcium imaging setup.
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FOOTNOTES |
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This work was supported by the National Heart, Lung, and Blood Institute Grant R29-HL-51130.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: D. H. Damon, Dept. of Pharmacology, Given Bldg., Rm. B-319, Univ. of Vermont, Burlington, VT 05405 (E-mail: ddamon{at}zoo.uvm.edu).
Received 20 January 1999; accepted in final form 8 September 1999.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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1.
Alberts, G. F.,
K. A. Peifly,
A. Johns,
J. F. Kleha,
and
J. A. Winkles.
Constitutive endothelin-1 overexpression promotes smooth muscle cell proliferation via an external autocrine loop.
J. Biol. Chem.
269:
10112-10118,
1994
2.
Antonelli-Orlidge, A.,
K. B. Saunders,
S. R. Smith,
and
P. A. D'Amore.
An activated form of transforming growth factor is produced by cocultures of endothelial cells and pericytes.
Proc. Natl. Acad. Sci. USA
86:
4544-4548,
1989
3.
Ardinger, H. H.,
R. H. Ardinger,
G. I. Bell,
and
J. C. Murray.
RFLP for the human transforming growth factor beta-1 gene (TGFB) on chromosome 19.
Nucleic Acids Res.
16:
8202,
1988
4.
Bevan, R. D.
Effect of sympathetic denervation on smooth muscle proliferation in the growing rabbit ear artery.
Circ. Res.
37:
14-19,
1975
5.
Blaes, N.,
and
J.-P. Boissel.
Growth-stimulating effect of catecholamines on rat aortic smooth muscle cells in culture.
J. Cell. Physiol.
116:
167-172,
1983[ISI][Medline].
6.
Chelmicka-Schorr, E.,
M. E. Checinski,
and
B. G. W. Arnason.
PC12 pheochromocytoma and sympathetic nervous derived trophic factors augment growth of neuroblastoma.
Eur. J. Cancer Clin. Oncol.
25:
1057-1059,
1989[ISI][Medline].
7.
Chirgwin, J. M.,
A. E. Przybyla,
R. J. MacDonald,
and
W. J. Rutter.
Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease.
Biochemistry
18:
5294-5299,
1979[Medline].
8.
Chun, L. Y.,
and
P. H. Patterson.
Role of NGF in the development of rat sympathetic neurons in vitro. I. Survival, growth and differentiation of catecholamine production.
J. Cell Biol.
75:
694-704,
1977
9.
Damon, D. H.
Postganglionic sympathetic neurons express endothelin.
Am. J. Physiol. Regulatory Integrative Comp. Physiol.
274:
R873-R878,
1998
10.
Damon, D. H.,
D. L. Lange,
and
B. G. Hattler.
In vitro and in vivo vascular actions of basic fibroblast growth factor (bFGF) in normotensive and spontaneously hypertensive rats.
J. Cardiovasc. Pharmacol.
30:
278-284,
1997[ISI][Medline].
11.
Dobbertin, A.,
P. Schmid,
M. Gelman,
J. Glowinski,
and
M. Mallat.
Neurons promote macrophage proliferation by producing transforming growth factor-2.
J. Neurosci.
17:
5305-5315,
1997
12.
Fingerle, J.,
K. H. Sanders,
and
Z. Fotev.
1-Receptor antagonists urapidil and prazosin inhibit neointima formation in rat carotid artery induced by balloon catheter injury.
Basic Res. Cardiol.
86, Suppl. 1:
75-81,
1991.
13.
Fort, P.,
L. Marty,
M. Piechaczyk,
S. El Sabrouty,
C. Dani,
P. Jeanteur,
and
J. M. Blanchard.
Various rat adult tissues express only one major mRNA species from the glyceraldehyde-3-phosphate-dehydrogenase multigenic family.
Nucleic Acids Res.
13:
1431-1442,
1985
14.
Haberstroh, U.,
G. Zahner,
M. Disser,
F. Thaiss,
G. Wolf,
and
R. A. K. Stahl.
TGF- stimulates rat mesangial cell proliferation in culture: role of PDGF -receptor expression.
Am. J. Physiol. Renal Fluid Electrolyte Physiol.
264:
F199-F205,
1993
15.
Hahn, A. W. A.,
T. J. Resink,
T. Scott-Burden,
J. Powell,
Y. Dohi,
and
F. R. Buhler.
Stimulation of endothelin mRNA and secretion in rat vascular smooth muscle cells: a novel autocrine function.
Cell Regul.
1:
649-659,
1990[ISI][Medline].
16.
Hamet, P.,
V. Hadrava,
U. Kruppa,
and
J. Tremblay.
Transforming growth factor 1 expression and effect in aortic smooth muscle cells from spontaneously hypertensive rats.
Hypertension
17:
896-901,
1991
17.
Lee, R. M. K. W.,
C. R. Triggle,
D. W. T. Cheung,
and
M. D. Coughlin.
Structural and functional consequence of neonatal sympathectomy on the blood vessels of spontaneously hypertensive rats.
Hypertension
10:
328-338,
1987
18.
Miller, D. A.,
A. Lee,
R. W. Pelton,
E. Y. Chen,
H. L. Moses,
and
R. Derynck.
Murine transforming growth factor 2 cDNA sequence and expression in adult tissues and embryos.
Mol. Endocrinol.
3:
1108-1114,
1989[Abstract].
19.
Nakaki, T.,
M. Nakayama,
S. Yamamoto,
and
R. Kato.
1-Adrenergic stimulation and 2-adrenergic inhibition of DNA synthesis in vascular smooth muscle cells.
Mol. Pharmacol.
37:
30-36,
1990[Abstract].
20.
Pelton, R. W.,
B. Saxena,
M. Jones,
H. L. Moses,
and
L. I. Gold.
Immunohistochemical localization of TGF1, TGF2, and TGF3 in the mouse embryo: expression patterns suggest multiple roles during embryonic development.
J. Cell Biol.
115:
1091-1105,
1991
21.
Ross, R.
The smooth muscle cell. II. Growth of smooth muscle in culture and formation of elastic fibers.
J. Cell Biol.
50:
172-186,
1971
22.
Schwartz, S. M.,
R. L. Heimark,
and
M. W. Majesky.
Developmental mechanisms underlying pathology of arteries.
Physiol. Rev.
70:
1177-1209,
1990
23.
Sherline, P.,
and
R. Mascardo.
Catecholamines are mitogenic in 3T3 and bovine aortic endothelial cells.
J. Clin. Invest.
74:
483-487,
1984.
24.
Stassen, F. R. M.,
N. J. H. Raat,
D. L. Brouwers-Ceiler,
G. E. Fazzi,
J. F. M. Smits,
and
J. G. R. De Mey.
Angiotensin II induces media hypertrophy and hyperreactivity in mesenteric but not epigastric small arteries of the rat.
J. Vasc. Res.
34:
289-297,
1997[ISI][Medline].
25.
Stouffer, G. A.,
and
G. K. Owens.
TGF-beta promotes proliferation of cultured SMC via both PDGF-AA-dependent and PDGF-AA-independent mechanisms.
J. Clin. Invest.
93:
2048-2055,
1994.
26.
Unsicker, K.,
K. C. Flanders,
D. S. Cissel,
R. Lafyatis,
and
M. B. Sporn.
Transforming growth factor beta isoforms in the adult rat central and peripheral nervous system.
Neuroscience
44:
613-625,
1991[ISI][Medline].
27.
Voyta, J. C.,
D. P. Via,
C. E. Butterfield,
and
B. R. Zetter.
Identification and isolation of endothelial cells based on their increased uptake of acetylated low density lipoprotein.
J. Cell Biol.
99:
2034-2040,
1984
28.
Wang, D.-J.,
N.-N. Huang,
and
L. A. Heppel.
Extracellular ATP and ADP stimulate proliferation of porcine aortic smooth muscle cells.
J. Cell. Physiol.
153:
221-233,
1992[ISI][Medline].
29.
Weber, H.,
M. L. Webb,
R. Serafino,
D. S. Taylor,
S. Moreland,
J. Norman,
and
C. J. Molloy.
Endothelin-1 and angiotensin-II stimulate delayed mitogenesis in cultured rat aortic smooth muscle cells: evidence for common signaling mechanisms.
Mol. Endocrinol.
8:
148-158,
1994[Abstract].
30.
Wolf, Y. G.,
L. M. Rasmussen,
and
E. Ruohslati.
Antibodies against transforming growth factor-1 suppress intimal hyperplasia in a rat model.
J. Clin. Invest.
93:
1172-1178,
1994.
31.
Zettler, C.,
R. J. Head,
and
R. A. Rush.
Chronic nerve growth factor treatment of normotensive rats.
Brain Res.
538:
251-262,
1991[ISI][Medline].
32.
Zukowska-Grojec, Z.,
P. Pruszczyk,
C. Colton,
J. Yao,
G. H. Shen,
A. K. Myers,
and
C. Wahlestedt.
Mitogenic effect of neuropeptide Y in rat vascular smooth muscle cells.
Peptides
14:
263-268,
1993[ISI][Medline].
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