From the Institut National de la Santé et de la Recherche
Médicale (INSERM) U337, Paris, France (Y.B., P.L., S.L.); and the
Department of Anatomy and Developmental Biology, University College London, UK
(G.G.).
Correspondence to Stéphane Laurent, Service de Pharmacologie, Hôpital Broussais, 96 rue Didot, Paris 75014, France. E-mail stephane.laurent{at}brs.ap-hop-paris.fr
Abstract
Abstract—We have recently
demonstrated that in large arteries of spontaneously hypertensive rats
(SHR), there is no increase of stiffness despite the increase in wall
thickness, a sign of mechanical adaptation of the arterial
wall to the higher level of stress. Because the dense plaques of smooth
muscle are a major site of anchorage between the muscle cells and
extracellular matrix, we determined by electron microscopy the
distribution of dense plaques and their connections to elastic lamellae
in the abdominal aorta of 1-year-old SHR and control Wistar rats. In
vivo echo-tracking measurement of aortic distensibility and elastic
modulus indicates a reduction of arterial stiffness in SHR
compared with Wistar rats when they are studied over a common range of
blood pressure. The media thickness to body weight ratio was higher in
SHR than in Wistar rats. In the media, the percentage of sectional area
occupied by extracellular matrix was not different between Wistar rats
and SHR. The average number of dense plaques per muscle cell was not
different between Wistar rats and SHR. However, the percentage of cell
surface occupied by dense plaques was increased in SHR, and the
percentage of cell surface connected to the elastic lamellae was twice
as high in SHR compared with Wistar rats (9.4±1.5% versus
3.8±1.1%). These results suggest that the elastin network plays a
major role in the mechanical adaptation of the arterial
wall in SHR, not through variations of its total amount but through
variations of the extent of anchorage to the muscle cells.
Until recently, it
was generally accepted that hypertension produces an increase in large
artery stiffness.1 2 3 4 However, our group and
others have shown that arterial stiffness is not increased
in hypertensive patients or in spontaneously hypertensive rats (SHR)
despite wall hypertrophy.5 6 7 8 9 A
recent study has shown that the aortic wall of SHR and Wistar rats has
similar mechanical properties despite the fact that in SHR the wall is
submitted to a higher level of stress.10 This
finding suggests that sustained hypertension is associated with a
restructuring of the arterial wall that produces the
mechanical adaptation of the arterial wall.
Electron microscopy has shown that the membrane-associated dense
plaques of muscle cells are a major site of anchorage of contractile
apparatus to extracellular matrix in
arteries.11 12 The mechanical link between muscle
cells and elastic lamellae provided by the dense plaques may play an
important role in regulating contractile and elastic tension in
mechanically stressed vessels. We hypothesized that the mechanical
adaptation of the arterial wall to hypertension involves an
increase in the links between muscle cells and elastic lamellae. Thus,
our objective was to measure the dense plaques and their connections to
elastic lamellae in a large elastic artery, the abdominal aorta, in
normal Wistar rats and SHR.
Methods
Aortic Mechanical Parameters
In Situ Perfusion and Fixation for Microscopy
Results
Arterial Mechanics
Mean circumferential wall stress calculated from in vivo
measurements of blood pressure and arterial diameter and in
vitro measurement of medial cross-sectional area after fixation at MAP
was increased in SHR compared with Wistar rats.
Structure of Abdominal Aortic Wall
Distribution of Membrane-Associated Dense Plaques
Discussion
The aim of the present study was to correlate mechanical
properties and ultrastructure of the abdominal aorta in hypertensive
and control rats. The main finding is a marked increase in connections
of muscle cell to elastic lamellae in hypertension.
As we recently showed,10 the incremental elastic
modulus of the aortic wall material (Einc),
determined for a given level of blood pressure, was not significantly
increased in SHR compared with Wistar rats. This indicates that the
arterial stiffness is not increased in SHR. However, the
circumferential wall stress is higher in SHR than in Wistar rats. Under
these conditions, an important issue is to determine how the
hypertensive artery is mechanically adapted despite the higher level of
stress. Considering that weight of SHR was lower than that of Wistar
rats, the increases in arterial thickness associated with
cellular hypertrophy and extracellular matrix accumulation
were only observed after normalization by body weight. We previously
found no difference between SHR and Wistar rats concerning elastin and
collagen densities. Because these compounds play a major role in the
mechanical properties of the arterial wall, we hypothesized
that hypertension induces some structural reorganization of the media,
namely, changes in the muscle to stroma connections, leading to
mechanical adaptation. Clark and Glagov,18 among
others, have shown that in the aorta of rabbit and pig,
membrane-associated dense bodies represent the mechanical
attachment site between elastic fibers and contractile
apparatus. These focal attachments are stable and have
great mechanical strength because they remain unchanged after
hyperdistension of the vessel.18 19 Here we have
shown that while some structural parameters are unchanged
in the hypertensive abdominal aorta (eg, density of elastin and
collagen, number of cells, and number of dense plaques per cell), there
is a significant increase in the percentage of the muscle cell surface
that is occupied by dense plaques. In contrast to our results, a study
on renovascular hypertension (2-kidney, 1-clip) showed a reduction of
the density of cell to elastic fiber contacts in hypertensive compared
with normotensive rats.20 This finding, however,
is not in disagreement with our hypothesis because in this model,
carotid stiffness was not adapted but increased compared with that in
normotensive animals.21 Thus, we would conclude
that the number of cell to elastic connections within the aortic media
is related to arterial wall stiffness. Another study of
hypertensive rats, which reported a decrease of membrane dense
bodies,19 is not directly comparable with ours
because it was carried out on mesenteric arteries.
The rise in blood pressure causes an increase in wall stress and an
enhancement of the stretch to which the stroma of the wall is
subjected. The stress is discharged via the dense plaques onto the cell
membrane, the cytoskeleton, and the contractile apparatus.
We suggest that it is this stress-induced activation of the muscle cell
that causes enhanced synthesis and secretion of
fibronectin10 and possibly of other extracellular
matrix components,22 as well as modifications in
cytosol composition (calcium and kinases) and in the contractile
apparatus and cytoskeleton.12 Among
the latter changes, we have now shown that there is the formation of
additional anchorage points of the cell to elastic lamellae. This
ultrastructural change alters the distribution of the wall stress onto
the muscle cells. We suggest that it also modifies the reciprocal
influences of the active and passive components of the wall, and thus
it contributes to the maintenance of the normal level of wall
stiffness despite the increase in stress.
In conclusion, the present study shows a novel organization of the
aortic media of SHR, characterized by changes in smooth muscle
cell–elastic lamellae connections, which may be a means for preserving
mechanical integrity of the arterial wall at elevated
arterial pressures. Ultrastructural characterization of
cell-matrix interactions could provide new insights into the mechanisms
by which genetic hypertension autoregulates the mechanical properties
of the arterial wall.
Acknowledgments
This study was supported by a grant from the Institut National
de la Santé et de la Recherche Médicale (INSERM No.
494014). We thank Bernadette Lucet and Mike Corder for excellent
technical assistance.
Received January 24, 1998;
first decision February 11, 1998;
accepted April 8, 1998.
References
1.
Cox R. Basis of the altered arterial
wall mechanics in the spontaneously hypertensive rat.
Hypertension. 1981;3:485–495.
2.
Dobrin P. Vascular mechanics. In: Shepherd JTAF, ed.
Handbook of Physiology. Vol 3. Baltimore, Md: American
Physiological Society; 1983:65–102.
3.
Levy B, El Fartak L, Pieddeloup C, Barouki F, Safar M.
Role of the endothelium in the mechanical response of
the carotid wall to calcium blockade in spontaneously hypertensive and
Wistar-Kyoto rats. J Hypertens. 1993;11:57–63.[Medline]
[Order article via Infotrieve]
4.
Safar M, Frohlich E. The arterial system
in hypertension: a prospective view. Hypertension. 1995;26:10–14.
5.
Laurent S, Hayoz D, Trazzi S, Boutouyrie P, Waeber B,
Omboni S, Brunner H, Mancia G, Safar M. Isobaric compliance of the
radial artery is increased in patients with essential hypertension.
J Hypertens. 1993;11:89–98.[Medline]
[Order article via Infotrieve]
6.
Laurent S, Girerd X, Mourad J, Lacolley P, Beck L,
Boutouyrie P, Mignot J, Safar M. Elastic modulus of the radial artery
wall material is not increased in patients with essential hypertension.
Arterioscler Thromb. 1994;14:1223–1231.
7.
Laurent S. Arterial wall
hypertrophy and stiffness in essential hypertensive
patients. Hypertension. 1995;26:355–362.
8.
Lacolley P, Ghodsi N, Glaser E, Challande P, Brissac
A, Safar M, Laurent S. Influence of graded changes in vasomotor tone on
the carotid arterial mechanics in live spontaneously
hypertensive rats. Br J Pharmacol. 1995;115:1235–1244.[Medline]
[Order article via Infotrieve]
9.
Hayoz D, Rutschmann B, Perret F, Niederberger M, Tardy
Y, Mooser V, Nussberger J, Waeber B, Brunner H. Conduit artery
compliance and distensibility are not necessarily reduced in
hypertension. Hypertension. 1992;20:1–6.
10.
Bézie Y, Daniel Lamazière J, Laurent S,
Challande P, Sa Cunha R, Bonnet J, Lacolley P. Fibronectin expression
and aortic wall elastic modulus in spontaneously hypertensive rats.
Arterioscler Thromb Vasc Biol. 1998. In press.
11.
Gabella G. Structural apparatus for force
transmission in smooth muscles. Physiol Rev. 1984;64:455–477.
12.
Small J, North A. Architecture of the smooth muscle
cell. In: Schwartz RM, ed. The Vascular Smooth Muscle Cell.
New York, NY: Academic Press Inc; 1995:169–188.
13.
Tardy Y, Hayoz D, Mignot J, Richard P, Brunner H,
Meister J. Dynamic noninvasive measurements of arterial
diameter and wall thickness. J Hypertens.
1992;10(suppl 6):S105–S109.
14.
Lacolley P, Glaser E, Challande P, Boutouyrie P, Mignot
J, Duriez M, Levy B, Safar M, Laurent S. Structural changes and in situ
aortic pressure-diameter relationship in long-term
chemical-sympathectomized rats. Am J Physiol. 1995;269:H407–H416.
15.
Lacolley P, Bezie Y, Girerd X, Challande P, Benetos A,
Boutouyrie P, Ghodsi N, Lucet B, Azoui R, Laurent S. Aortic
distensibility and structural changes in sinoaortic denervated rats.
Hypertension. 1995;26:337–340.
16.
Gabella G. Complex structure of the common carotid
artery of sheep. Anat Rec. 1995;243:376–383.[Medline]
[Order article via Infotrieve]
17.
Gabella G. Asymmetric distribution of dense bands in
muscle cells of mammalian arterioles. J Ultrastruct Res. 1983;84:24–33.[Medline]
[Order article via Infotrieve]
18.
Clark J, Glagov S. Structural integration of the
arterial wall, I: relationships and attachments of medial
smooth muscle cells in normally distended and hyperdistended aortas.
Lab Invest. 1979;40:587–602.[Medline]
[Order article via Infotrieve]
19.
McGuffee L, Little S. Tunica media remodeling in
mesenteric arteries of hypertensive rats. Anat Rec. 1996;246:279–292.[Medline]
[Order article via Infotrieve]
20.
Sosa-Melgarejo J, Berry C. Cell to stroma contacts in
the tunica media of the hypertensive rat thoracic aorta. Arch Med
Res. 1996;27:123–126.[Medline]
[Order article via Infotrieve]
21.
Zanchi A, Wiesel P, Aubert J, Brunner H, Hayoz D. Time
course changes of mechanical properties of the carotid artery in renal
hypertensive rats. Hypertension. 1997;29:1199–1203.
22.
Ito H. Vascular connective tissue changes in
hypertension. In: Lee R, ed. Blood Vessel Changes in
Hypertension: Structure and Function, Vol I. Boca Raton, Fla: CRC
Press; 1989:99–122.
© 1998 American Heart Association, Inc.
Third Workshop on Structure and Function of Large
Arteries: Part I
Connection of Smooth Muscle Cells to Elastic Lamellae in Aorta of Spontaneously Hypertensive Rats
Key Words: aorta • muscle, smooth • rats, inbred SHR • elastin
We simultaneously recorded arterial
diameter and blood pressure at the abdominal aorta in pentobarbital
anesthetized rats to determine the pressure-diameter and
pressure-distensibility curves, as previously
described.5 6 9 13 14 15 The incremental elastic
modulus (Einc) was calculated from in vivo
distensibility and in vitro measurement of wall cross-sectional area
after fixation at mean arterial pressure (MAP).
Arterial diameter measurement was obtained using an
ultrasonic echo-tracking device (NIUS-01, Asulab SA).
Segments of abdominal aorta were obtained from 1-year-old male
Wistar rats (n=3) and SHR (n=3) weighing 632±36 g and 408±32 g,
respectively. The abdominal aorta was fixed in situ at MAP by perfusion
via the heart using a fixative consisting of 4%
glutaraldehyde and 1% paraformaldehyde
in 0.1 mol/L sodium cacodylate buffer (pH 7.4).16
The specimens were then postfixed in 1% osmium tetroxide in cacodylate
buffer for 1 hour, washed in several changes of distilled water, and
immersed in a saturated solution of uranyl acetate for 1 hour. They
were then dehydrated in ethanol and epoxy-propane and placed in
araldite for infiltration. Semithin sections (0.5 µm) were cut
either transversely to the length of the vessel (for area and shape of
the lumen and for area and thickness of the media) or longitudinally,
ie, parallel to the length of the vessel and through its middle (for
thickness of the wall and for study of muscle cells and stroma). These
sections were stained with toluidine blue and observed with a Zeiss
photomicroscope with phase-contrast optics. Ultrathin sections (100 nm)
were cut longitudinally, collected on copper grids, stained with uranyl
acetate and lead citrate, and viewed in a Philips 400 electron
microscope. For quantification, photographic montages were made,
reconstructing the full thickness of the wall for a length of 150 to
400 µm at a magnification of x3700. Dense plaques were
identified by the presence of electron-dense areas on the cytoplasmic
side of the cell membrane.17 Measurements were
made using a video imaging technique (Quancoul)10
to calculate the transverse sectional area of nucleated cell profiles,
the cell surface to cell perimeter ratio, the percentage of
extracellular space in the media, the number and lengths of dense
plaques, and the proportion of dense plaques connected to elastic
lamellae.16
Table 1
shows the mechanical
parameters of abdominal aorta in SHR and Wistar rats. At
MAP, which reflects the operational pressure level, distensibility was
lower and Einc higher in SHR than in Wistar rats.
The distensibility-pressure curve of SHR was shifted to the right
compared with that of Wistar rats (data not shown), indicating that
arterial distensibility was not decreased in SHR for a
given level of blood pressure.8 9 Thus, the lower
value of aortic distensibility at MAP, under
physiological conditions, was explained by the
higher level of blood pressure.
View this table:
[in a new window]
Table 1. Individual Values of Mechanical Parameters of
Abdominal Aorta in 1-Year-Old SHR and Control Wistar
Rats
The abdominal aorta fixed in situ at MAP was circular in profile,
and its internal radius measured 595±18 and 520±50 µm in
Wistar rats and SHR, respectively. Media thickness was 50.4±3.3 and
47.5±3.0 µm in Wistar rats and SHR, respectively. The media
thickness to body weight ratio was higher in SHR than in Wistar rats
(116±11 versus 80±3 µm/kg). In both strains, the media was
made of elastic lamellae alternating with layers of muscle cells. The
extracellular space ranged between 40% and 50% in the media and was
not different between Wistar rats and SHR. Each layer of muscle was 1
cell thick, and most of the cells ran approximately circumferentially.
No difference in cell size was apparent between the 2 strains of rats,
and the area of nucleated cell profiles ranged from 21 to 42
µm2 in both. The cell profiles in transverse
sections were irregular in shape and contour, but without any obvious
differences between the 2 strains studied. The cell profiles
presented deep invaginations, laminar or fingerlike (in the
latter case, they often appeared isolated from the cell surface)
(Figure). The cell membrane at the cell surface proper and in the
invaginations was in contact with basal lamina and extracellular
materials. For the present purposes, we recognized two
configurations, namely, the cell membrane formed 2 distinct domains:
(1) some areas of the membrane were in contact with a basal lamina, and
immediately beyond, with microfibrils and collagen fibrils, and (2)
other areas of the membrane were in contact with elastic fibers and
elastic lamellae. Elastic fibers and elastic lamellae were connected
with each other, and a precise separation was difficult. However, it
was still possible to distinguish, within the elastin-adhesion domains
of the membrane, areas of direct contact with elastic lamellae and
areas of contact with elastic fibers, and we considered for
quantification only those in direct contact with elastic lamellae.
View larger version (135K):
[in a new window]
Figure 1. Muscle cell attachment to elastic lamellae. The cytoplasm
along the plasma membrane that borders the elastin shows areas of dense
plaques. The percentage of dense plaques connected to elastic lamellae
is increased in SHR (A) compared with Wistar rats (B). Original
magnification x20 000.
Dense plaques were studied in longitudinal sections of the
vessels. All the muscle cells of the media had prominent dense plaques
encrusting the cell membrane and projecting deep into the cell.
Smooth muscle cell to elastic lamina connections were defined as
elastin expansions that spanned obliquely from the elastic laminae to
the surface of the smooth muscle cell, where they attached in a region
occupied by membrane-associated dense plaque (Figure
). The number of
total dense plaques per cell was not different between Wistar rats and
SHR (12.0±0.4 versus 11.3±0.4). However, the percentage of cell
surface occupied by dense plaques was increased in SHR compared with
Wistar rats (42±3% versus 34±2%). The percentage of
dense bands connected to elastic laminae was increased 2-fold in SHR
compared with that in Wistar rats (9.4±1.5% versus 3.8±1.1%; Table 2).
View this table:
[in a new window]
Table 2. Distribution of Membrane-Associated Dense Plaques
and Their Connections to Elastic Lamellae in 1-Year-Old SHR and Control
Wistar Rats
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