© 1995 American Heart Association, Inc.
Articles |
Differential Effects of Pressure and Flow on DNA and Protein Synthesis and on Fibronectin Expression by Arteries in a Novel Organ Culture System
From the Institut National de la Santé et de la Recherche Médicale, Unité 141 (N.B., G.J.K., R.M., A.T.) and Unité 127 (J.-L.S.), Institut Fédératif de Recherche "Circulation Lariboisière," Paris, France.
Correspondence to Alain Tedgui, PhD, INSERM Unité 141, 41 Boulevard de la Chapelle, 75010 Paris, France.
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Abstract |
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Abstract Structural adaptation of the blood vessel wall occurs in response to mechanical factors related to blood pressure and flow. To elucidate the relative roles of pressure, flow, and medium composition, we have developed a novel organ culture system in which rabbit thoracic aorta, held at in vivo length, can be perfused and pressurized at independently varied flow and pressure for several days. Histology and histomorphometry, as well as scanning electron microscopy, revealed a well-preserved wall structure. In arteries perfused and pressurized at 80 mm Hg, endothelial injury led to a 2-fold increase in [3H]thymidine incorporation in the media, which peaked at 3 to 5 days and returned to baseline level at 6 to 8 days. In intact endothelialized vessels cultured for 3 days under no-flow conditions, pressure per se had no effect on DNA synthesis. In contrast, in the presence of serum, total protein synthesis, as assessed by [35S]methionine incorporation into the media, was enhanced 6-fold at 150 mm Hg compared with vessels pressurized at 0 or 80 mm Hg. In intact vessels perfused at a constant flow of 40 mL/min for 3 days, DNA synthesis was unchanged regardless of the pressure level when vessels were cultured in the presence of serum but increased 8-fold at both 80 and 150 mm Hg in the absence of serum. Unlike DNA synthesis, total protein synthesis was enhanced 12-fold by flow regardless of the presence or absence of serum. Expression of fibronectin was markedly enhanced at high transmural pressure, and serum potentiated its expression in the arterial wall. This novel organ culture system of perfused and pressurized vessels allowed identification of differential effects of pressure, flow, and serum on DNA and total protein synthesis, including cellular fibronectin expression.
Key Words: pressure • flow • artery • organ culture • fibronectin
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Introduction |
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Mechanical forces regulate cell growth and biosynthesis in many tissues.1 In arteries and veins, mechanical forces related to pressure and flow are crucial in determining blood vessel wall adaptation in normal and diseased states.2 3 4 This has been shown by using, for example, in vivo models in which animals were made hypertensive5 6 7 8 or in which blood flow in a single vessel was experimentally increased or decreased.9 10 11 12 Vascular remodeling may have important clinical implications during the evolution of several vascular diseases. Vascular remodeling may alter compliance in hypertension and atherosclerosis,13 cause vascular fragility and compensatory changes in atherosclerosis,14 and lead to restenosis after angioplasty.15 Understanding of the modeling-remodeling processes is particularly critical for developing successful therapies in vascular pathology.
Although in vivo models have proved useful in establishing a relation between vascular remodeling and mechanical forces (ie, tensile stress and wall shear stress) to which blood vessels are subjected, they do not facilitate the understanding of mechanogenic transduction properties at the cellular and molecular levels, because they do not allow distinction between neurohormonal and mechanical effects. Therefore, the in vitro cell culture model has been extensively used and has permitted identification of factors that might affect smooth muscle and endothelial cell proliferation and biosynthesis.16 Although several models for mechanically stimulating cultured cells have been developed,17 18 19 this approach has certain limitations. The in vivo micro-environment differs markedly from the in vitro culture conditions; the behavior of cultured cells is not identical to that in vivo. Smooth muscle cells in culture lose their contractile phenotype and assume a synthetic phenotype.20 21 Endothelial–smooth muscle cell interactions and cell-matrix interactions that could be critical in modulating the cell response are not reproduced. An alternative approach has been to use organ culture of vessels.22 23 24 25 26 27 28 29 30 31 In those models, however, vessels were not pressurized or perfused. In an attempt to elucidate the specific effects of mechanical forces on the blood vessel wall and to determine how mechanical signals are transduced to generate a biochemical response, we devised a novel organ culture model in which rabbit thoracic aorta could be perfused and pressurized at independently varied flow and pressure under controlled conditions of intravascular and extravascular medium composition. The aim of the present study was to demonstrate the long-term viability of the rabbit aortic vessel wall in this new model of organ culture and to examine the effects of pressure, flow, and serum on DNA and total protein synthesis after 3 days of culture as well as on the expression of c-Fn.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Artery Preparation
Male New Zealand White rabbits (2 to 2.5 kg) were anesthetized with sodium pentobarbital (30 mg · kg-1 IV). The animals were intubated and mechanically ventilated. The abdominal and pleural cavities were opened, and the entire length of the descending thoracic aorta was exposed. The artery was dissected free from the isthmus to the abdominal aorta, and the intercostal arteries were cauterized 2 to 3 mm from the vessel, which was cleaned of adhering periadventitial tissue. A cannula (external diameter, 3.25 mm; internal diameter, 2.15 mm) fitted to the internal diameter of the aorta was inserted retrogradely into the distal end of the aorta and connected to a reservoir placed 100 cm above the animal. The proximal end of the aorta was then ligated just below the arch, and a second cannula, pointing distally, was inserted anterogradely. By use of this procedure, pressure was maintained continuously within the artery, preventing collapse of the vessel wall and endothelial damage. A ligature was tied around the midregion of the aorta, and a cannula was inserted distal to this ligature. The lower part of the descending thoracic aorta was then excised on splints while held at in vivo length and under physiological pressure in order to preserve the structural and metabolic integrity of the vessel wall, as previously shown by silver nitrate staining of endothelial junctions and histoenzymatic determination of phosphorylase activity.32 33 A similar procedure was used to excise the upper part of the aorta.
The surgical preparation of arterial segments for subsequent organ culture was carried out under sterile conditions. Aortic segments were immersed in an organ culture bath placed in an incubator and filled with DMEM (GIBCO BRL) containing antibiotics (penicillin, 100 IU · L-1; streptomycin, 100 mg · L-1; and amphotericin B, 10 µg · L-1) supplemented with 20% decomplemented FCS (Boehringer Mannheim France SA) or 1.5% bovine serum albumin (Sigma Chemical Co).
Organ Culture
To vary the transmural pressure and the flow independently, each
arterial segment was connected to a perfusion circuit (Fig 1
) consisting of a custom-designed and -constructed
three-port glass reservoir, peristaltic pump (Masterflex 60648,
Cole-Palmer Instrument Co), and pressure chamber. The upper port of the
glass reservoir was connected to the pressure chamber, which permitted
the application of controlled hydrostatic pressure to the intraluminal
compartment. The two lateral ports of the glass reservoir were used for
the input and the output of the circulating intraluminal medium, which
was the same as the extraluminal medium described above. The
peristaltic pump was connected between the distal end of the aortic
segment and the upper lateral part of the glass reservoir. Arteries
were pressurized at 80 or 150 mm Hg and maintained either under
no-flow conditions or perfused at a constant flow of 40 mL/min with
the same medium culture as described above. Control experiments were
carried out in vessels maintained under relaxed conditions at zero
transmural pressure and zero flow.
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In addition, to assess the effect of serum, vessels were incubated either in the presence of 20% FCS in culture medium or in serum-free medium supplemented with 1.5% albumin. Vessels were kept at 37°C in an incubator under humidified air gassed with 5% CO2 for periods of time up to 8 days. The medium was changed every 3 days. In a series of sequential interval experiments, we established that biochemical parameters (pH, glucose and ion contents, PO2, and PCO2) of the culture media were unchanged.
Assessment of Vascular Wall Integrity
DNA Content and Histomorphometry
To evaluate the preservation of vessel wall composition, total
DNA, elastin, and collagen contents were determined in a series of
experiments at intervals up to 8 days after removal from the living
animal.
For each arterial segment, an arterial ring was removed, the media/intima was separated from adventitia, and length and weight were determined. The tissues were dried for 12 hours at 50°C in a glass well. Total DNA content in the intima/media was measured by DABA fluorescence assay.34 A 100 µL solution containing 32 mg DABA was added. After 45 minutes of incubation at 55°C, 3 mL HCl at 1 mol/L was added to the well, and fluorescence was measured by using Salmon sperm DNA (Sigma) as a standard.
A ring of each cultured segment was fixed in a 2% solution of
paraformaldehyde and embedded in paraffin. Transverse
cross sections (5 µm) of the aorta were stained with
hematoxylin-eosin-safran, orcein, and Sirius red and Masson's
trichrome stains. Slides were processed with an automatic
image-analysis processor (NS 1500, Nachet-Vision). Orcein
and Sirius red staining permitted determination of elastin and collagen
content, respectively. Elastin and collagen densities were measured in
12 locations spaced evenly around the perimeter of the vessel, and a
mean value was calculated for each section. Cross-sectional area
was measured by using computer-assisted planimetry with the same
image-analysis processor, which permitted calculation of
total elastin and collagen contents. We have found in earlier studies a
highly significant correlation between rat aortic collagen content when
using biochemical (hydroxyproline determination35 after
collagen isolation using the method of Lansing et al36 as
modified by Wolinsky5 ) and histomorphometric
analysis (Fig 2). On the other hand, using data
reported by Michel et al,37 who determined rat aortic
elastin content by biochemical analysis and morphometric
methods identical to those used in the present work, we obtained a
highly significant correlation between the two methods:
y=2.58+0.94x, r=.87, n=7,
P<.01.
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Scanning electron microscopy and immunohistochemical studies were performed in 3- and 8-day cultured vessels as well as in freshly removed aortas.
Electron Microscopic Studies
Pressurized vessels were flushed with, and relaxed vessels were
incubated in, 0.1 mol/L PBS, pH 7.4, containing 5%
glutaraldehyde and 4% formaldehyde, with transmural
pressure held constant. Fixative was also placed in the external bath.
The vessels were left to fix for 180 minutes. Three aortic rings were
obtained from each vessel for scanning electron microscopy.
Endothelial morphology was studied by scanning electron
microscopy. Samples for study were dehydrated in a gradient series of
ethanol and acetone solutions and critical point–dried by using
five flushes of liquid CO2. Three pieces of dried tissue
from each arterial segment (each 
5 mm2),
corresponding to the proximal, medial, and distal part of the vessel,
were mounted on aluminum stubs, sputter-coated with gold, and
viewed in a Jeol JSM T200 scanning electron microscope.
Immunocytochemical Studies
Arterial rings were frozen in isopentane precooled
with liquid nitrogen and stored at -80°C. 
-Smooth muscle actin, a
marker of contractile smooth muscle phenotype, and vWF and ACE,
specific for endothelial cells, were detected in
unfixed cryosections by using mouse anti-human -smooth
muscle actin antibodies (1:100, Dako S.A.), rabbit
anti-human vWF polyclonal antibodies (1:200, Dako S.A.), and
mouse anti-ACE monoclonal antibodies (generous gift from Dr S. Danilov,
INSERM U36, Paris).38
DNA and Total Protein Synthesis
In an attempt to assess the responsiveness of the organ culture
model, the time course of 3H-TdR incorporation (for
measurement of DNA synthesis) was studied in intact and
deendothelialized vessels that were perfused at
40 mL/min, cultured in the presence of serum, and pressurized at 80
mm Hg or maintained under relaxed (no-flow and no-pressure)
conditions for periods of time up to 8 days.
Deendothelialization was performed by mechanical
removal of the endothelium. After complete excision of
the aorta, a 2F embolectomy catheter was inflated with
physiological saline serum and gently passed into
the vessel under visual control. This procedure was repeated three
times in order to ensure a complete endothelial
denudation without medial injury, as assessed by optical microscopy.
Using the same procedures for vessel excision and
endothelial removal, we previously showed by
transmission electron microscopy that the internal elastic lamina and
the medial layer were totally preserved.39
On the basis of the observation that DNA synthesis in deendothelialized vessels peaked by 3 to 5 days, a culture period of 3 days was chosen to determine the effect of flow, pressure, and serum on DNA, total protein synthesis, and fibronectin expression in intact endothelialized vessels.
DNA and total protein synthesis were determined by the incorporation of 3H-TdR and 35S-Met into DNA and protein, respectively.40 For the last 6 hours of the incubation period, 3H-TdR (2 µCi/mL; specific activity, 5 Ci/mmol; Amersham France S.A.) and 35S-Met (2 µCi/mL; specific activity, >1000 Ci/mmol; Amersham France S.A.) were added to the intraluminal and extraluminal media. The aortic segments were then removed from the cannulas, rinsed four times in ice-cold PBS for 5 minutes, stripped of the adventitia, and weighed. Intimal/medial preparations were homogenized in PBS, and tissue proteins were precipitated with cold TCA (final concentration, 10%). After centrifugation, the precipitate was suspended in 5% TCA and heated in a water bath at 90°C for 20 minutes to hydrolyze and extract DNA and to remove tRNA-bound 35S-Met. After a second centrifugation, the supernatant containing DNA was counted by liquid scintillation spectrometry, and the pellet was washed successively with cold 5% TCA and ethanol. TCA-insoluble material was solubilized overnight at 37°C in NaOH (1N) and then counted by liquid scintillation spectrometry. The results were expressed as disintegrations per minute per milligram wet tissue.
As a control of specificity of the pressure and flow effects on protein synthesis, cycloheximide (20 µg/mL) was added to the culture medium supplemented with or without FCS.
To localize cells incorporating 3H-TdR and 35S-Met, autoradiographs were performed in 3-day cultured vessels incubated separately in the presence of 3H-TdR or 35S-Met and studied under two experimental conditions (relaxed and perfused at high pressure without serum), in which a large difference was observed in 3H-TdR and 35S-Met incorporation. Three independent experiments were performed in each experimental condition. Frozen arterial cross sections were coated with NTB2 nuclear track emulsion (Eastman Kodak) and exposed for 21 and 14 days at 4°C to 3H and 35S, respectively. Sections were then developed, poststained with hematoxylin and eosin, fixed, washed in distilled water, and dehydrated through alcohol to xylene. The labeled cells were examined in light microscopy in four serial cross sections of each vessel. The total number and the number of labeled nuclei were counted. Adventitial cells were excluded from this analysis. A minimum of six areas from the same cross section were quantified, giving one mean value. 3H-TdR indexes were obtained by dividing the number of silver-stained nuclei by the total number of nuclei.
Fibronectin Expression
c-FN expression was detected in arterial cross
cryosections (5 µm), which were first incubated overnight at 4°C in
mouse anti-human c-FN monoclonal antibodies (Sigma) at a dilution
of 1:200. These antibodies have been shown to recognize only cellular
and not plasma fibronectin.41 Sections were then
postincubated for 30 minutes at 37°C in anti-mouse–specific
biotin-conjugated antibodies (1:200, Amersham plc) and amplified
by the streptavidin–Texas red system (1:50, Amersham plc) for 30
minutes at room temperature. Fluorescence staining was
visualized with a Leitz microscope equipped with an
epifluorescence system (Leica).
Quantitative analysis of thoracic aorta labelings with antibodies against c-FN was performed with a video-imaging microscopy technique. Video images from a low-light-level camera (C-2400, Hamamatsu) were transmitted to a microcomputer (Macintosh IIfx) equipped with an image-analyzer program (OPTILAB, Graphtek). This software permitted us to store several images from the same cross section in digitally calibrated formats. The image-analyzer software automatically evaluated the positive labelings by using a threshold method, ie, selecting pixels whose intensity level was greater than a threshold value (background). All quantitative analyses were performed in a blinded fashion. Data are the mean of three independent experiments in each experimental condition. For each aortic fragment, a minimum of four sections were quantified, giving one mean value. Quantitative analysis was expressed as percent total aortic surface occupied by c-FN.
Statistical Analysis
Results are expressed as mean±SEM. To evaluate the effect of
deendothelialization on DNA synthesis, ANCOVA was
performed with culture time as covariable and
endothelium and pressure levels used as independent
factors. A two-way ANOVA was constructed with 3H-TdR
incorporation and 35S-Met incorporation data to test the
effect of pressure, flow, and serum. Comparisons were performed by use
of Bonferroni's test. Differences were considered significant at
P<.05.
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Arterial Wall Integrity
Light microscopy was used to investigate the integrity of the arterial wall just after preparation of the vessels and before culture (Fig 3A
) as well as after different times
of culture (Fig 3). Up to 8 days of culture, the media was well
preserved, and the histological structure was
indistinguishable from that of freshly isolated aortic segments. Smooth
muscle cells were normally oriented, with no evidence of cellular
swelling or necrosis. The medial layer, defined by the presence of
20 concentric elastic fiber layers, contained smooth muscle cells
disposed in a manner characteristic of the normal rabbit thoracic
aorta. Noninjured arteries showed a morphologically intact and
continuous endothelial monolayer.
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To evaluate the preservation of vessel wall composition in cultured
arteries, total DNA content and elastin and collagen densities were
determined in a series of experiments at intervals up to 8 days after
removal from the living animal in the presence of a constant flow
(Tables 1 through 3).
Except for an initial small decrease of <10% in total DNA content
(statistically not significant), no further alteration in tissue
composition was evident regardless of the experimental conditions of
pressure.
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When studied by scanning electron microscopy, the luminal surface of
cultured vessels under the different experimental conditions (pressure,
flow, and serum) presented a largely undamaged and normal
endothelial structure with slightly protruding
endothelial nuclei (Fig 4). In 3-day
arteries fixed at 80 or 150 mm Hg (Fig 4B and 4C), intact and
stretched endothelial cells, oriented in the direction
of the flow, were observed. In 8-day vessels fixed at 150 mm Hg (Fig 4D), the endothelial monolayer was preserved intact,
and ovoid bulging of endothelial nuclei could be seen.
In freshly excised vessels (Fig 4A), fixed under relaxed conditions,
the luminal surface presented a typical smooth layer covering
coarse longitudinal folds. We did not observe any difference in the
endothelial structure in vessels cultured with or
without serum.
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-Smooth muscle actin, characteristic of the contractile
phenotype, remained normally expressed in smooth muscle cells
of 3-day cultured arteries regardless of the pressure level (80 or 150
mm Hg) (Fig 5B and 5C) and was not affected by the
experimental conditions of flow or serum. In 8-day cultured and
pressurized vessels, even though -smooth muscle actin was still
detected in smooth muscle cells (Fig 5D), a slight decrease in
-smooth muscle actin expression was observed regardless of the
pressure level (80 or 150 mm Hg). The only observed modification of
the vascular structure associated with the culture conditions was the
stretching of smooth muscle cells and elastic fibers and the decrease
in wall thickness due to high pressure imposed for 3 to 8 days. In the
opposite condition, smooth muscle cells and elastic fibers were curled
up when vessels were cultured in the relaxed state (zero transmural
pressure, zero flow).
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The two endothelial markers (vWF and ACE) were detected
at the level of endothelial cells regardless of the
experimental conditions of flow and medium composition and regardless
of the pressure level (80 or 150 mm Hg) (Fig 6). A
similar pattern of vWF and ACE was observed in 3- and 8-day cultured
arteries.
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Effect of Deendothelialization on
3H-TdR Incorporation
To assess the responsiveness of the organ culture model, we
evaluated the time course (up to 8 days) of DNA synthesis after
endothelium removal in vessels cultured in the presence
of serum, perfused at 40 mL/min, and pressurized at 80 mm Hg compared
with vessels maintained in culture under no-flow, no-pressure
conditions. An increase in 3H-TdR incorporation was found
only in deendothelialized, perfused, and
pressurized arteries (n=10, P<.006) but not in
deendothelialized relaxed arteries (n=6,
P=.96). Moreover, ANCOVA revealed a significant effect of
culture time in 80 mm Hg–pressurized and perfused arteries
compared with intact arteries (n=10, P<.05). In
deendothelialized and pressurized arteries,
this increase in 3H-TdR incorporation peaked at days 3 to 5
and returned to baseline level at days 6 to 8 (Fig 7).
The maximum value of 3H-TdR incorporation in
deendothelialized arteries was about twofold
higher than the values in intact arteries, which did not vary
significantly with time.
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Light microscopy did not show any neointimal proliferation in cultured arteries even after deendothelialization.
Effects of Pressure and Flow on DNA and Total Protein
Synthesis
Inasmuch as DNA synthesis peaked at 3 to 5 days, the differential
effects of pressure, flow, and serum on total DNA and protein synthesis
were studied in vessels cultured for 3 days. Protein synthesis was
measured by 35S-Met incorporation during the last 6 hours
of culture. The specific effect of the transmural pressure was
evaluated in vessels maintained in culture under no-flow
conditions. Pressure had no effect on DNA synthesis either in the
presence of FCS (1.14±0.8x10-3,
0.55±0.24x10-3, and 0.87±0.04x10-3
dpm/mg wet tissue at 0, 80, and 150 mm Hg, respectively) or in the
absence of FCS (0.58±0.22x10-3,
1.19±0.28x10-3, and 1.01±0.33x10-3
dpm/mg wet tissue at 0, 80, and 150 mm Hg, respectively).
Conversely, total protein synthesis was markedly enhanced by fivefold
to sixfold at 150 mm Hg in the presence of serum
(5.62±0.87x10-3 dpm/mg wet tissue) compared with relaxed
conditions (0.77±0.2x10-3 dpm/mg wet tissue) or with
vessels pressurized at 80 mm Hg (1.22±0.52x10-3 dpm/mg
wet tissue). In the absence of serum, total protein synthesis was
unaffected by transmural pressure
(0.70±0.25x10-3,
1.22±0.52x10-3, and 0.92±0.26x10-3
dpm/mg wet tissue at 0, 80, and 150 mm Hg, respectively).
When vessels were perfused at 40 mL/min with culture medium containing
FCS, DNA synthesis was not stimulated regardless of the level of
transmural pressure (Fig 8A). However, in the absence of
FCS, DNA synthesis was markedly increased by 8-fold at both 80 and 150
mm Hg (8.12±0.69x10-3 and 8.58±1.42x10-3
dpm/mg wet tissue, respectively) compared with relaxed conditions
(0.58±0.22x10-3 dpm/mg wet tissue). Unlike DNA
synthesis, total protein synthesis was enhanced 12-fold by flow
regardless of the presence or absence of serum (Fig 8B). As a control
of the specificity of the pressure and flow effects on protein
synthesis, cycloheximide added to culture medium for 3 days clearly
inhibited the increase in 35S-Met incorporation whatever
the experimental conditions (Table 4).
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Autoradiographs of transverse cross sections were used to locate the
3H-TdR– and 35S-Met–labeled cells in
relaxed vessels and perfused arteries pressurized at 150 mm Hg in the
absence of serum. In relaxed aortas, replication of smooth muscle cells
was very low (<0.1%) (Fig 9A) compared with cells in
pressurized arteries (1.7±0.2%) (Fig 9B). Proliferating cells were
located in outer layers of the media. Autoradiographs performed to
locate cells incorporating 35S-Met showed almost
undetectable labeled cells in relaxed vessels (Fig 9C), whereas all
smooth muscle cells were positive in vessels pressurized at high
pressure (Fig 9D). No 35S-labeled cell was detected in the
adventitia.
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Effect of Pressure and Flow on c-Fn Expression
When c-Fn expression was analyzed throughout the
arterial wall, an increase in c-FN was seen at high
transmural pressure (Fig 10). In the absence of serum,
c-FN expression was only detected at the luminal side of the vessel
wall when vessels were kept at 0 or 80 mm Hg (Fig 10A) and occupied
5.78±0.5% or 6.3±0.9% of the aortic wall, respectively (Fig 11). At 150 mm Hg, c-FN expression was increased
throughout the media and occupied 11.1±0.7% of the aortic wall
(P<.05), with immunostaining being positive
in the innermost layers of the media (Fig 10B). FCS did not affect the
c-FN expression in vessels maintained at 0 or 80 mm Hg (5.4±0.5% or
5.6±0.5%, respectively) (Fig 10C) but amplified it at high transmural
pressure (21.5±2.7%, versus 11.1±0.7% in vessels cultured at 150
mm Hg without serum, P<.001) (Fig 10D).
Immunostaining was positive in the whole inner third of
the media of vessels pressurized at 150 mm Hg and cultured in medium
containing 20% FCS.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Arteries in their physiological environment are permanently exposed to arterial pressure and blood flow that engender mechanical forces (tensile stress and wall shear stress) acting on the vessel wall. Vessels can adapt to any change in these mechanical factors. Elucidation of the mechanisms of pressure- and/or flow-induced vascular remodeling is therefore an important issue in vascular biology under normal and diseased states.3
The first concern of the present study was to develop a novel organ culture model in which pressure and flow could be varied independently in vessel segments and in which the composition of the culture medium could be carefully controlled.
The findings of the present study are summarized in Table 5 and indicate the following: (1) Rabbit aortic segments
can be maintained in organ culture under conditions of controlled flow
and pressure for as long as 8 days after removal from the living
animal. (2) Pressure per se is capable of enhancing total protein
synthesis in the presence of serum but has no effect on DNA synthesis.
(3) Flow increases DNA synthesis in serum-free conditions and
stimulates total protein synthesis regardless of the presence or
absence of serum. (4) Fibronectin expression in the media is markedly
enhanced by high transmural pressure.
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To the best of our knowledge, the present model is the first to allow the possibility of independent change in pressure and flow in arteries maintained in culture for several days. Earlier studies have been performed in organ culture models of vessels to overcome limitations encountered in cell cultures.22 23 24 25 26 27 28 29 30 31 However, in these studies vessels were cultured in a relaxed and undistended state, in the absence of flow and pressure, with the aim being mainly to investigate the mechanisms of intimal hyperplasia as seen in vivo after endothelium removal. Recently, Mangiarua et al42 developed an organ culture model in which rat aortic segments were perfused under constant pressure level in order to evaluate the effect of culture on the contractile response of vascular smooth muscle cells. However, despite the presence of an unchanged tunica media, the intimal surface was mostly devoid of endothelial cells. Therefore, morphological and functional characteristics of the aortic segments were modified during the perfusion period under these conditions. Furthermore, this model did not allow independent change in flow and pressure. Kuo et al43 have developed a system in which vessels were perfused at various flow rates and under different pressure levels by using two reservoirs, one for inflow and the other for outflow. The two reservoirs were moved in the same direction to generate intraluminal pressure and simultaneously moved in opposite directions to generate a pressure gradient–driven flow. This dual open-reservoir system was used only for short-term studies (a few hours) and would not permit long-term culture.
In deendothelialized cultured arteries maintained at 80 mm Hg, tritiated labeling revealed a significant increase in DNA synthesis, which supports the viability of the artery segments. However, no intimal hyperplasia was observed in the present study, in contrast to what is usually seen in vivo.44 45 Deendothelialization with an intra-arterial embolectomy catheter usually induces a necrotizing injury of the underlying media. Medial damage has been shown to be necessary to cause smooth muscle cell proliferation in in vivo animal models of arterial injury.46 In the present study, we performed a gentle denudation without evidence of medial injury. Gentle denudation applied to vessels in our model could explain the histological absence of neointimal proliferation observed in other studies of deendothelialized arteries. It is noteworthy that this DNA synthesis depended on the presence of pressure and flow. An increase in 3H-TdR incorporation was found only in deendothelialized, perfused, and pressurized arteries but not in relaxed arteries at zero transmural pressure and under no-flow conditions. This observation underlines the importance of mechanical factors as mediators of vascular remodeling as well as the potential value of our system for controlling the level of flow and pressure to explore the effects of these mechanical factors over a wide range. It has been shown that relaxed rat renal arteries cultured in vitro exhibit smooth muscle cell proliferation after deendothelialization.47 Paradoxically, however, endothelial cells have been observed to promote proliferation of arterial smooth muscle cells in several organ culture models of the relaxed vessel wall,28 29 48 whereas endothelial abrasion diminished DNA synthesis.31 49 50 The contrasting responses of smooth muscle cells to endothelial denudation in relaxed organ culture could be due to the absence of mechanical stress.
In the present study, no significant changes in total DNA content and elastin and collagen contents were found in the media of intact arteries maintained under conditions of controlled pressure and flow. However, although the relatively short periods of observation in our studies did not permit the study of slow structural changes involving elastin or collagen synthesis, our perfused and pressurized organ culture model did permit the study of DNA and protein synthesis and of specific protein expression in relation to variations of pressure and wall shear.
Effects of Pressure on DNA and Total Protein
Synthesis
The effects of pressure on both DNA and total protein synthesis
have been studied in various animal models of hypertension. Vascular
smooth muscle cells are capable of a variety of morphological responses
within the blood vessels of hypertensive animals. The
arterial wall could grow as a result of cell
hypertrophy, in association with increased protein
synthesis. It could also expand its synthetic capacity by increasing
the DNA content via cell polyploidy or
hyperplasia.16 51 52 However, the exact role of mechanical
(pressure) versus humoral factors in vascular remodeling seen in
hypertension is still unclear.
To investigate the proper effect of transmural pressure on DNA and total protein synthesis, vessels were maintained in culture for 3 days under no-flow conditions and were studied either at zero transmural pressure or at 80 or 150 mm Hg. Our finding that pressure per se had no effect on DNA synthesis regardless of the presence or absence of serum is in agreement with a recent report by Holycross et al,53 who showed that DNA synthesis in intact rat aortic rings was not stimulated by 13% fetal bovine serum, in contrast to previous studies by De Mey et al26 and Schiffers et al.54 Holycross et al discussed the possibility that this discrepancy might be due to the fact that endothelial cells were not removed in their experiment. Leung and colleagues17 18 also found no change in DNA synthesis in a model of cultured smooth muscle cells subjected to cyclic stretching. In contrast, we did find an effect of pressure per se on total protein synthesis in the presence of serum. Only high transmural pressure stimulated 35S-Met incorporation. In their work, Leung et al also observed that the application of stretch to smooth muscle cells in culture resulted in a marked increase in protein synthesis, particularly in collagen. Holycross et al did not find any change in protein synthesis in aortic rings mounted on steel support and subjected to a low load of 1.5 g for 16 hours, which is consistent with our finding that protein synthesis was not stimulated at low pressure (80 mm Hg). It is noteworthy that FCS was required at 150 mm Hg to enhance protein synthesis. Thus, elevated pressure triggered protein synthesis, but some unknown factors were required for full biological response. In contrast to this result, Grande et al55 found a significant increase in protein and collagen synthesis only when cyclically stretched smooth muscle cells were cultured in serum-free medium. Differences between cell and organ culture models might account for this discrepancy.
Effects of Flow on DNA and Total Protein Synthesis
The role of flow on vascular remodeling in vivo is well
documented.9 10 11 56 57 Langille and
O'Donnell9 demonstrated that persistent decrease in flow
led to an endothelium-dependent reduction of the
luminal diameter with structural adaptations, including decrease in
elastin content in the immature rabbit.10 57 Conversely,
increased blood flow produced by an arteriovenous
fistula11 resulted in enlargement of vessel caliber, in
such a way that wall shear stress was normalized. These authors
proposed the existence of a local autoregulatory mechanism of wall
shear stress involving protein turnover in the arterial
wall.
To account for the flow-induced adaptation of the arterial wall dependent on endothelial cells, the use of in vitro cell culture systems to investigate the effect of flow on vascular endothelial cells has been widely developed in the last decade (see reviews in References 58 and 5958 59 ), but hitherto, no system allowing long-term investigations of the effect of flow on the whole intact arterial wall has been available.
The present model permits us to show that DNA and total protein synthesis can be stimulated by flow regardless of the pressure level. However, whereas flow-induced protein synthesis was not affected by the presence or absence of serum, perfused cultured vessels exhibited increased DNA synthesis only in serum-free conditions. These results suggested either that the absence of serum favored the production of mitogens, possibly of endothelial origin, in response to flow or that the flow-induced release of these mitogens was inactivated in the presence of serum. It has been shown that endothelial cells in culture subjected to fluid shear stress are capable of producing platelet-derived growth factor60 and endothelin,61 62 which are known to possess mitogenic activity.63 64 65 The fact that total protein synthesis was stimulated by flow regardless of the presence or absence of serum while increased DNA synthesis by flow was observed only in serum-free conditions suggested that the factors involved in flow-induced protein synthesis might differ from those responsible for flow-induced DNA synthesis.
Effect of Pressure on Fibronectin Expression
Fibronectin is a dimeric cell-adhesive extracellular matrix
glycoprotein secreted by mesenchymal cells. This protein is
capable of interacting with macromolecules, including fibrin and
collagen, as well as bearing specific fibronectin receptors on their
surfaces. Fibronectin has multiple biological functions in
embryogenesis and tissue repair, including cell attachment, migration,
proliferation, and cytodifferentiation.66 It might
influence the smooth muscle cell response in
hypertension.67 68 At 150 mm Hg, a marked expression of
c-Fn was observed in the innermost smooth muscle cell layers of the
media, and serum potentiated the pressure effect and revealed
fibronectin accumulation in the whole inner third of the media. Because
the anti-fibronectin antibody used in the present study did not
recognize the plasma form of fibronectin,41 this observed
increase in fibronectin in the media cannot be due to increased influx
of fibronectin from serum as a result of a high pressure–induced
increase in endothelial permeability. It might be
accounted for by increased de novo synthesis and/or reduction of
extracellular fibronectin. However, this latter possibility is
unlikely, since quiescent adult smooth muscle cells do not express c-Fn
as do fetal or dedifferentiated smooth muscle cells. Our results
showing that fibronectin expression was markedly increased at high
pressure are consistent with those reported by Takasaki et
al69 showing in vivo that elevation in blood pressure
increased fibronectin expression in the rat aorta.
Consistent with our findings, it has been demonstrated that cardiac myocytes grown on silicone membrane undergo increased protein synthesis and expression of specific genes in response to passive stretch.70 However, the signal transduction pathways of mechanical stress remain to be elucidated. Interestingly, in cardiac myocytes, stretch activates multiple second-messenger systems71 72 and causes autocrine release of angiotensin II, which acts as initial mediator of stretch-induced protein synthesis.73 Likewise, an autocrine vascular renin-angiotensin system might be involved in pressure-induced protein synthesis.74
We have described a new model of perfused and pressurized rabbit thoracic aorta in an organ culture, which permits the investigation of mechanisms leading to smooth muscle cell proliferation in the whole artery. Effects of mechanical factors such as pressure and flow on vascular remodeling can be easily reproduced, including independent variations of pressure and flow such as occur in the living animal.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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Nathalie Bardy holds a Glaxo Laboratories graduate studentship. The authors thank Drs D. Chiarasini and V. Vergès-Belmin for expert advice with the scanning electron microscopy studies.
Received August 4, 1994; accepted July 7, 1995.
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References |
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K. G. Birukov, S. Lehoux, A. A. Birukova, R. Merval, V. A. Tkachuk, and A. Tedgui Increased Pressure Induces Sustained Protein Kinase C–Independent Herbimycin A–Sensitive Activation of Extracellular Signal–Related Kinase 1/2 in the Rabbit Aorta in Organ Culture Circ. Res., December 19, 1997; 81(6): 895 - 903. [Abstract] [Full Text]
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F. Pourageaud and J. G. R. De Mey Structural properties of rat mesenteric small arteries after 4-wk exposure to elevated or reduced blood flow Am J Physiol Heart Circ Physiol, October 1, 1997; 273(4): H1699 - H1706. [Abstract] [Full Text] [PDF]
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S. P. Allen, S. S. Wade, and R. L. Prewitt Myogenic Tone Attenuates Pressure-Induced Gene Expression in Isolated Small Arteries Hypertension, August 1, 1997; 30(2): 203 - 208. [Abstract] [Full Text]
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A. Sabri, B. I. Levy, P. Poitevin, L. Caputo, E. Faggin, F. Marotte, L. Rappaport, and J. L. Samuel Differential Roles of AT1 and AT2 Receptor Subtypes in Vascular Trophic and Phenotypic Changes in Response to Stimulation With Angiotensin II Arterioscler. Thromb. Vasc. Biol., February 1, 1997; 17(2): 257 - 264. [Abstract] [Full Text]
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G. Meyer, R. Merval, and A. Tedgui Effects of Pressure-Induced Stretch and Convection on Low-Density Lipoprotein and Albumin Uptake in the Rabbit Aortic Wall Circ. Res., September 1, 1996; 79(3): 532 - 540. [Abstract] [Full Text]
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N. Bardy, R. Merval, J. Benessiano, J.-L. Samuel, and A. Tedgui Pressure and Angiotensin II Synergistically Induce Aortic Fibronectin Expression in Organ Culture Model of Rabbit Aorta: Evidence for a Pressure-Induced Tissue Renin-Angiotensin System Circ. Res., July 1, 1996; 79(1): 70 - 78. [Abstract] [Full Text]
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H. Zhang and J. E. Faber Trophic Effect of Norepinephrine on Arterial Intima-Media and Adventitia Is Augmented by Injury and Mediated by Different {alpha}1-Adrenoceptor Subtypes Circ. Res., October 26, 2001; 89(9): 815 - 822. [Abstract] [Full Text] [PDF]
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