© 1999 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
Atrophic Remodeling of the Artery-Cuffed Artery
From the Toronto Hospital Research Institute (I.M.B., B.L.L.); the Samuel Lunenfeld Research Institute (S.L.A.), Mount Sinai Hospital; and the Departments of Laboratory Medicine and Pathobiology (I.M.B., B.L.L.), Physiology (I.M.B., S.L.A.), and Obstetrics and Gynecology (S.L.A., B.L.L.), University of Toronto, Toronto, Ontario, Canada.
Correspondence to B. Lowell Langille, CCRW 1-836, The Toronto Hospital (General Division), 200 Elizabeth St, Toronto, Ontario, M5G 2C4, Canada. E-mail lowell.langille{at}utoronto.ca
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
Abstract—Increased arterial wall tension stimulates growth and remodeling of arteries, but little is known about the effects of decreased wall tension, despite its developmental and pathological significance. Consequently, we cuffed 1 carotid artery in rabbits with a portion of the contralateral artery to off-load circumferential wall tension. The model produced rapid and extensive atrophy of the cuffed artery that yielded decreases in the DNA content of the cuffed artery (a measure of cell number) from 8.0±0.5 µg/cm of in situ vessel length to 5.6±0.5 µg/cm at 21 days postoperatively. The elastin content of the cuffed artery was also significantly reduced, from 399±17 to 283±17 µg/cm, and collagen content was reduced from 468.0±59.0 to 154±24 µg/cm (P<0.05) at 21 days postoperatively. Detection of DNA oligonucleosomes by gel electrophoresis implicated apoptotic cell death in remodeling due to cuffing. Upregulation of matrix metalloproteinases (MMPs), including MMP-2, MMP-9, and unidentified gelatinases, indicated that these enzymes may also be involved in remodeling. No further changes in wall structure were seen between 3 weeks and 6 months, and the excised artery that was used as a cuff exhibited normal medial morphology for at least 6 months postoperatively. We infer from these experiments that off-loading of arterial wall tension induces rapid and extensive atrophy of the arterial media.
Key Words: apoptosis • extracellular matrix • matrix metalloproteinase • wall tension
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Long-term changes in local hemodynamic forces induce remodeling of arterial wall tissue that is critical to vascular development, to vascular adaptation in the adult, and to the progression of cardiovascular disease.1 This remodeling is specific for the type of hemodynamic load that is imposed on the tissue: pressure-induced increases in tension elicit thickening of the arterial wall, whereas flow-induced increases in shear stress result in increased vessel diameter. Furthermore, these remodeling processes are age dependent. For example, increases or decreases in blood flow rate have a marked effect on tissue accumulation in immature arteries, whereas flow-induced changes in the diameters of mature arteries are achieved with little, if any, net changes in wall constituents,2 unless flow is greatly increased for a prolonged time.3
With changes in blood flow rate, the most basic features of tissue reorganization and its time course depend on whether flow is increased or decreased. For example, remodeling in response to decreased blood flow normally proceeds much more rapidly than does remodeling elicited by increased flow.2 3 By contrast, only very limited comparisons have been made between the effects of increased versus decreased arterial blood pressure and wall tensile stress. Instead, investigations have concentrated on the effects of increased pressure and tensile stress because of the putative importance of the resulting arterial wall thickening to the progression of hypertension.4 However, remodeling in response to decreased arterial pressure is physiologically significant, because it occurs in some vessels during development, eg, the pulmonary arterial system after birth5 ; furthermore, clinically significant arterial occlusive disease depressurizes the arterial tree downstream from the occlusion site. Consequent remodeling may affect both progression of the disease and the sequelae to therapeutic interventions such as bypass graft implantation. Finally, there is evidence that experimental off-loading of tensile stress inhibits experimental atherogenesis,6 so even the initiation of arterial disease may be affected.
Recently, we cuffed rabbit abdominal aortas with rigid polyethylene collars to investigate the effect of reduced vessel wall distension on intimal proliferative responses to endothelium-denuding (balloon) injury.7 These intimal responses were largely unaffected, but the media of the arteries atrophied and displayed apoptosis and histological evidence of disruption of matrix, with or without an injury stimulus.
In the current study, we have characterized the atrophic responses to the reduced wall distension by using a modified cuffing procedure. Instead of placing synthetic cuffs on aortas, we cuffed rabbit carotid arteries with segments of the contralateral carotid artery to preclude the effects of reactions to foreign materials. Cuffed arteries were assessed at up to 6 months after surgery. We found that the transplanted carotid arteries that were used as cuffs remained viable and displayed normal medial structure at all time points. In contrast, the cuffed artery, like the cuffed aorta, exhibited extensive atrophy. Apoptosis yielded significant cell loss by 3 weeks (30% decrease in DNA content); furthermore, matrix degradation resulted in substantial decreases in elastin (29%) and collagen (67%) contents. A dramatic upregulation of expression of matrix metalloproteinases (MMPs) was consistent with a role for these enzymes in matrix degradation.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Experimental Alteration of Arterial Wall Tension
Full surgical anesthesia of adult, male New Zealand White rabbits weighing 3.0 to 4.9 kg was induced with an intramuscular injection of 0.8 mL/kg of xylazine (20 mg/mL) and ketamine (90 mg/mL). Anesthesia was maintained with a continuous intravenous infusion (0.031 mL/min) of this anesthetic.
The carotid arteries were exposed via a midline cervical incision, and the left common carotid artery was cleared of adventitia under a dissecting microscope. The right common carotid artery was then cleared of adventitia, twice ligated as far proximally and distally as possible, and then excised between the ligations and opened lengthwise. The right carotid artery was sewn, by using a 7-0 prolene suture, as a cuff around the left carotid artery. The neck incision was closed in 2 layers, and the animals were allowed to recover.
Immediately after surgery; at 5, 10, or 21 days (n=6 per time period) after surgery; or 6 months (n=3) postoperatively, rabbits were killed with an intravenous injection of 1.0 mL of euthanasia solution: 200 mg/mL N-[2-(m-methoxyphenyl)-2-ethybutyl(1)]-2-hydroxybutyramide, 50 mg/mL 4,4'-methylene-bis-(cyclohexyltrimethylammonium iodide), and 5 mg/mL tetracaine hydrochloride (T-61, Hoechst Canada, Inc). After a rapid thoracotomy was performed, the descending thoracic aorta was retrogradely cannulated, and the carotid arteries were flushed with 60 mL of PBS and then fixed by perfusion with 3% paraformaldehyde in PBS for 20 minutes at 100 mm Hg via the aortic catheter. The cuffed segments of artery, as well as a segment of carotid artery upstream from the cuff, were excised and embedded in paraffin; then transverse or longitudinal sections were made and stained with hematoxylin and eosin or Movat's modified pentachrome connective-tissue stain.
Ligation of the right carotid artery before excision causes the left
carotid artery to carry increased (collateral) flow to cranial tissues.
We wished to test the effect of this flow increase on the remodeling we
observed in cuffed arteries; therefore, in an additional group of
rabbits (n=6), the external left carotid artery was ligated at the time
of cuffing. We have previously reported that this ligation reduces
ipsilateral common carotid artery flow rate by 
70%.2 8
In the current study, flow rates were determined by reanesthetizing the
rabbits 3 weeks after surgery and mounting transit-time ultrasonic flow
probes (Transonic Systems) on the experimental carotid artery, and
blood flow rates were recorded on a Gould 2400S chart recorder.
This work was approved by the Animal Care Committee of the
Toronto Hospital and was conducted in accord with the standards
of the Canadian Council on Animal Care.
Morphometry
Histological sections were examined using a
Nikon photomicroscope (Labophot) equipped with a Pulnix TMC-7 video
camera. The camera was linked to a computer imaging analysis
system (C• Imaging, model 640, Compix, Inc) that was used to measure
medial cross-sectional area as a measure of medial tissue mass, and
luminal circumference. The cuffed (left common carotid) artery segments
were compared at the different time points with control (unmanipulated)
carotid arteries.
Biochemical Analyses
Adventitia was removed from the cuffed and upstream segments of
carotid arteries 21 days postoperatively, and the segments were divided
into bilateral, longitudinal segments (n=6 rabbits). Elastin and
collagen contents were determined from 1/2 of the vessel while
the other half was used to determine DNA content. Two additional
rabbits were used for DNA analysis. Contents were expressed as
micrograms per centimeter of in situ vessel length.
DNA content was determined using the fluorometric assay of LaBarca and Paigen.9 In brief, arterial tissue was homogenized and reacted with the fluorescent dye bisbenzimide (Hoechst 33258, Sigma Chemical Co). Binding of DNA enhances fluorescence of the dye in proportion to DNA concentration. The DNA in the arterial samples was then determined by comparison with a standard curve generated by using varying amounts of calf thymus DNA type I (Sigma).
Collagen and insoluble elastin were measured after treatment of the arterial tissue with CNBr, a reagent that cleaves proteins at methionine residues. Elastin lacks methionine residues and remained insoluble after digestion. CNBr (50 mg/mL) was added to 70% formic acid that had been degassed by bubbling with N2. Arterial tissue samples were suspended in 1 mL of the solution in tightly capped tubes and incubated for 24 hours at room temperature. The elastin residue was removed from the solution, and the digestion was terminated by adding 10 mL of boiling distilled water; then the elastin residue was frozen and lyophilized.
A ninhydrin assay was used to determine the amount of elastin that was isolated by the above procedure. Insoluble elastin samples were hydrolyzed with 5.7N HCl at 110°C overnight and then dried at 130°C for 3 hours. Samples were reconstituted in distilled water. Standards ranging in concentration from 0 to 10 mg/mL were prepared using acid-hydrolyzed bovine ligamentum nuchae elastin (Sigma).10 Nin-Sol AF dimethyl sulfoxide–ninhydrin reagent (1 mL, Pierce) was added to 2 mL of standard or sample in glass test tubes, and the tubes were boiled in a water bath for 15 minutes. Absorbance of standards and samples was read on a spectrophotometer at 570 nm (LKB Ultraspec Plus, model 4054, Pharmacia, LKB, Biochrom), and the amount of elastin present in samples was determined by interpolation from the standard curve.10
Collagen content was measured by assaying the CNBr extract for hydroxyproline.11 In brief, the diluted CNBr extract was lyophilized, the residue was hydrolyzed overnight in 5.7N HCl at 110°C, and 4-hydroxyproline was determined in this hydrolysate by colorimetry.2 Because collagen is the only protein in the extract containing significant quantities of hydroxyproline, the total weight of collagen in the tissue was calculated from this hydroxyproline content, after assuming that collagen contains 12.77% 4-hydroxyproline by weight.12
Gelatin Zymography
Zymography was performed on protein extracted from carotid
arteries at 5, 10, and 21 days after cuffing (n=3 per time period).
Cuffed arteries were isolated, stripped of cuffs and adventitial
tissue, frozen in LN2, and ground to a fine
powder. Protein was extracted in lysis buffer containing 1% SDS and
100 µmol/L PMSF and 10 µg/mL leupeptin in 45 mmol/L Tris
buffer (pH 7.0). Protein concentration was determined using the
bicinchoninic acid protein assay reagent (Pierce). Equal amounts of
total protein from each extract were electrophoresed on a 10%
SDS-polyacrylamide gel that had been copolymerized with 0.1%
type I gelatin (Sigma) to provide a substrate for
collagenases/gelatinases. After electrophoresis, the gels
were washed with 2.5% Triton X-100 and then incubated overnight at
37°C in 0.05 mol/L Tris, 2.5 mmol/L CaCl2,
and 0.02% NaN3. Some gels were incubated in the
presence of 2 mmol/L 1,10-phenanthroline or 20 mmol/L EDTA to
inhibit metalloproteinase activity. Gels were stained with Coomassie
blue, and sites of gelatin degradation appeared as lytic bands in the
stained gel.
Western Immunoblotting
Zymograms displayed lytic bands at 85, 67, and 60 kDa. To test
whether these bands were produced by MMP-9 or MMP-2,
arterial protein extracts were electrophoresed as described
above and then transferred to nitrocellulose membranes (Protran). The
membranes were immunoblotted with either primary
anti–human MMP-9 (mouse) antibody (ICN) or anti–human MMP-2 (mouse)
antibody (ICN) diluted 1:2500, and enhanced chemiluminescence (ECL,
Amersham Life Science) was used to detect the immunoreactive bands.
After being blotted with peroxidase-labeled anti-mouse secondary
antibody, the membranes were blotted with detection reagent and then
exposed to Kodak X-OMAR x-ray film.
Detection of Degradation of DNA Into Oligonucleosomes
DNA was extracted from upstream and cuffed segments at 5, 10,
and 21 days postoperatively (n=5 per time period) and purified using a
standard extraction method. Tissues were minced and incubated overnight
in DNA lysis buffer (20 mmol/L Tris-HCl [pH 7.4], 1% SDS,
5 mmol/L EDTA, 100 µg/mL proteinase K, and 10 µL/mL RNase A)
at 50°C. The DNA was extracted with salt-saturated phenol and
chloroform and precipitated with 100% ethanol.13 Two
micrograms of arterial tissue DNA or 1 µg of 100-bp DNA
marker (Pharmacia Biotech) was incubated with 10 µCi of
[32P]dCTP (ICN Biomedical Canada Ltd) and 10 U
Klenow polymerase (Pharmacia Biotech) for 15 minutes at 30°C. DNA was
also incubated with 0.75 ng of a 30-bp oligonucleotide
to control for variability in loading wells for electrophoresis. The
end-labeling reaction was terminated by addition of 10 mmol/L
EDTA. Unincorporated nucleotides were removed by using
ProbeQuant G-50 Micro Columns (Pharmacia Biotech) and by following the
manufacturer's instructions. The end-labeled DNA was then
electrophoresed on a 1.8% agarose gel and blotted onto Hybond nylon
membranes (ICN Biomedical Canada Ltd), and the membranes were used to
expose Kodak X-OMAR x-ray film to detect the end-labeled DNA
fragments.
Statistical Analysis
All data are expressed as mean±SEM. Comparisons between cuffed
carotid artery medial cross-sectional areas and luminal circumferences
from different time points and with unmanipulated carotid arteries were
made using a 1-way ANOVA and Fisher's protected least significant
difference test. Comparisons of DNA, elastin, and collagen contents of
upstream and cuffed vessels were analyzed by paired
t tests. Differences were considered significant at
P<0.05.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Histology
Histology of the carotid artery upstream from the cuff is shown in Figure 1A
. This vessel segment was
identical to control carotid arteries harvested from unmanipulated
animals (data not shown). Matrix degradation in the cuffed artery,
evident as modest fragmentation of the elastic lamellae, was observed
as early as 5 days postoperatively (not shown). At 21 days, extensive
matrix degradation, cell loss, and attenuation of the media were
evident (Figure 1B). No further changes to the cuffed artery
were detected at 6 months postoperatively. Interestingly, the segment
of right carotid artery that was used as a cuff remained viable and
exhibited normal medial morphology after 21 days (Figure 1B) and
6 months, although some degeneration, dehiscence, and inflammatory cell
accumulation were observed around the suture site, but not
elsewhere.
|
Morphometry
Morphometry revealed marked atrophy of the cuffed artery over the
21 days after cuffing, as indicated by a decrease in medial
cross-sectional area from 0.28±0.01 to
0.17±0.02mm2 at 3 weeks (Figure 2A). The decreased cross-sectional area
was statistically significant by 5 days (P<0.05), and a
further significant decrease was detected at 3 weeks. Luminal
circumferences (Figure 2B) of cuffed segments were acutely
reduced by 37%, from 4.77±0.06 to 3.0±0.2 mm, and
circumferences remained significantly below control values
(P<0.05) at 0, 5, and 10 days postoperatively.
Circumferences returned to near-normal values at 21 days. Thinning of
the cuffed artery undoubtedly contributed to this restoration of
diameter, although dehiscence of the cuff margins was also probably a
factor at later time points.
|
Ligation of the right common carotid artery, which was necessary for
excision of the cuff segment, causes doubling of left common carotid
artery blood flow rate.14 15 To test whether this increase
in flow rate contributed to the remodeling produced by cuffing, we
ligated the left external carotid artery in a separate group of rabbits
at the time of cuffing. Three weeks later, blood flow rate in the left
common carotid artery (18.8±2.2 mL/min) was slightly below values that
we and others have recorded in control carotid arteries of
rabbits14 15 16 ; thus, the ligation procedure eliminated
flow rate increases associated with cuffing. The ligation did not
significantly influence vessel circumference or medial cross-sectional
area after cuffing (Figure 2
). We infer that alterations in flow
rate do not significantly contribute to remodeling associated with
cuffing.
Biochemical Analysis of Wall Constituents
Our observation that cuffing of the left carotid artery induced
medial tissue loss was supported by biochemical analysis of
wall tissue constituents (Figure 3). DNA
content of the cuffed artery was significantly lower than that of the
upstream segment of artery (5.6±0.5 compared with 8.0±0.5 µg/cm,
P<0.05) at 21 days.
|
Elastin content was also significantly lower at 21 days in cuffed arteries when compared with the upstream segments (283±17 compared with 399±17 µg/cm, P<0.05). Similarly, collagen content of the cuffed artery was reduced from 468.0±59.0 to 154±24 µg/cm (P<0.05) at 21 days postoperatively.
Gelatin Zymography
Gelatin zymography of protein isolated from unmanipulated control
carotid arteries revealed constitutive expression of 67- and 60-kDa
gelatinases in the rabbit carotid artery (Figure 4A). Expression of these enzymes was
enhanced in cuffed arterial segments at all time points. In
addition, gelatinolytic enzymes with molecular
masses of 108, 100, 85, 67, 60, and 75 kDa were induced in cuffed
arteries. Incubation of gels with 2 mmol/L 1,10-phenanthroline or
20 mmol/L EDTA completely inhibited all of the proteinases
detected (data not shown), suggesting that these
gelatinolytic enzymes were metalloproteinases.
|
Western Immunoblotting
Western immunoblotting was used to test whether
bands detected by zymography were produced by MMP-9 and/or MMP-2.
Anti–human MMP-2 antibody detected the 67- and 60-kDa gelatinases
(Figure 4B). Anti–human MMP-9 antibody detected the 85- and
75-kDa gelatinases as well as the bands detected by anti–MMP-2
antibody. We infer that the 67- and 60-kDa gelatinases are the latent
and active forms, respectively, of MMP-2 and that the 85- and 75-kDa
gelatinases are latent and active forms, respectively, of MMP-9.
Presumably, the anti–MMP-9 antibody cross-reacts with MMP-2 in
rabbits, as has been reported by the manufacturer for guinea pigs.
Detection of DNA Fragmented Into Oligonucleosomes
When DNA extracted from cuffed vessels at 5 days postoperatively
was end-labeled with [32P]dCTP and
electrophoresed on an agarose gel, DNA fragments in multiples of 200 bp
were detected (Figure 5). These data
indicate cleavage of DNA at internucleosomal sites by
endogenous endonucleases, a biochemical hallmark of
apoptosis. These same findings were also observed at 10 and 21
days postoperatively (data not shown). Degradation of DNA into
oligonucleosomes was observed as early as 2 days postoperatively, the
earliest time point studied. Some apoptosis was detected at low
levels in the upstream segments of the left carotid artery at early
time points. This may be due to a nonspecific response to surgical
manipulation of the artery.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Hemodynamic forces are very potent initiators of arterial remodeling. Chronic changes in blood flow rates lead to long-term alterations in arterial diameter, whereas increased blood pressure and wall tension cause arterial wall thickening. Although such remodeling has been extensively studied in the past, the primary focus has been on its growth-related aspects, ie, cell proliferation and matrix synthesis. In recent years, however, the importance of cell death and matrix degradation has received increasing emphasis in vascular biology.17 For example, we recently showed that while increased blood flow rate stimulates growth of developing arteries,15 suppression of blood flow not only inhibits cell proliferation, but it also enhances endothelial and smooth muscle cell apoptosis.8 Cell death is probably important not only in suppressing cell accumulation rates, but also it is probably involved in more subtle reorganization of wall tissue in remodeling arteries. In this regard, it is noteworthy that both the onset of hypertension and its reversal upregulate apoptosis.18 19 20 Similarly, matrix degradation is important in vascular pathologies, including atherosclerosis21 22 23 and restenosis,24 and matrix degradation is involved in vascular tissue responses to defined, experimental injury to arteries. It is also likely that local, tightly regulated matrix degradation is important in reshaping arteries that are exposed to changing mechanical loads. Consequently, experimental models that produce relatively high levels of cell death and matrix degradation may be especially valuable for studying these processes.
Because elevated blood pressure and wall tension induce substantial cell and matrix accumulation in arteries, we reasoned that off-loading wall tension might provide an effective stimulus for these atrophic processes. Furthermore, remodeling in response to reduced wall tension has important developmental and pathophysiological implications. For example, the pulmonary arterial system experiences a rapid and dramatic decrease in blood pressure in the immediate neonatal period, with pressures falling from 45 to 50 mm Hg to 10 to 20 mm Hg shortly after birth. The implications of this fall in blood pressure and pulmonary arterial wall tension for neonatal growth and remodeling of the pulmonary vasculature have been addressed only rarely.25 In addition, the clinical consequences of some of the most critical vascular disorders, eg, atherosclerosis and thrombosis, are a direct result of arterial obstruction that compromises blood flow. By definition, arterial obstructions that create enough flow resistance to compromise perfusion will decrease blood pressure, and therefore wall tension, in downstream arteries. Rapid atrophic remodeling at these sites may affect the sequelae to arterial occlusive disease. For example, extensive production of matrix-degrading enzymes adjacent to lesion sites may contribute to plaque rupture and embolism. Furthermore, such remodeling farther downstream could have important implications for the outcome of bypass grafts that are directed to these sites.
The artery-cuffed artery was designed to off-load wall tension in arteries without exposing the tissue under study to foreign materials. Furthermore, by using a long, straight, uniform artery, the carotid, we were able to locally off-load wall tension in 1 segment while leaving an adjacent, upstream segment undisturbed to serve as a control. Essentially, the cuffing procedure effectively doubles wall thickness (h) of the vessel and reduces vessel radius (R), so that the tensile stress (T) imposed by arterial pressure (P), ie, T=PxR/h, is substantially reduced. This net reduction in tension is experienced preferentially by the cuffed artery because of its smaller diameter and because additional tensile stress is imposed on the outer, cuffing artery when its cut edges are apposed during suturing.
The model produced the atrophic responses we predicted; however
we were struck by the potency and rapidity with which these occurred.
The vessel underwent extensive atrophy over 21 days, which comprised
loss of medial mass, smooth muscle cell apoptosis, and
extensive degradation of medial extracellular matrix; thus, significant
reductions in vessel wall DNA (30%), elastin (29%), and collagen
(67%) contents were observed. Zymography and Western blot
analysis indicated that atrophy was accompanied by marked
upregulation and activation of gelatinolytic
enzymes, including MMP-2 and MMP-9, plus other unidentified
gelatinases. The accumulated effects of these processes were a decrease
in medial tissue mass, as assessed by medial cross-sectional area, of
35%. The subsequent structural stability of the cuffed artery
between 3 weeks and 6 months indicates that the vessel reached
equilibrium with hemodynamic loads by the earlier time
point.
Our observations of rapid loss of matrix proteins, with the expression of a diverse family of matrix-degrading enzymes, indicate that reduced wall tension is a very potent stimulus for remodeling matrix, especially because these proteins are normally very stable.1 Zymography showed constitutive activity of 67- and 60-kDa gelatinases in unmanipulated control arteries, and Western immunoblots demonstrated that this activity is attributable to the latent and active forms of MMP-2, which is constitutively expressed in vascular tissue. (Latent enzymes produce lytic bands with zymography because SDS in the gels causes conformational changes in the protein that expose the active site.) Activity associated with MMP-2 was upregulated in cuffed arteries; in addition, off-loading wall tension induced expression of 5 more gelatinolytic enzymes with molecular masses of 108, 100, 85, 75, and 37 kDa. Western immunoblots indicated that the 85-kDa gelatinase was MMP-9. The antibody used in Western blots also detected the 67- and 60-kDa bands, but cross-reactivity of the antibody with MMP-2 has been reported previously, according to information provided by the supplier. The induction of MMP-9 during arterial remodeling is consistent with findings from other investigators.26 27 Its expression may be especially noteworthy, because arterial elastin is degraded by MMP-9.28 We have not yet identified the origins of other lytic bands on the zymograms.
Cell death was also extensive in cuffed arteries, and fragmentation of DNA extracted from cuffed arteries into oligonucleosomes indicated that cell death occurred via apoptosis. This mode of cell death plays an important role in developmental remodeling of large arteries13 as well as in a variety of vascular pathologies.29 30 31 32 In particular, reductions in flow rate through developing arteries substantially upregulate apoptosis in these vessels.8 In previous studies, we found that death or proliferation of endothelial cells rapidly restored cell density to resting levels when arteries remodel in response to altered hemodynamics,2 14 a finding that suggests that density-dependent signals control apoptosis and mitosis rates. It is possible that density-dependent signals also control medial smooth muscle cell populations in the cuffed arteries we examined; however, there are other possibilities. For example, tensile stresses regulate expression of mitogens, and many of these also are survival factors that protect against apoptosis. Thus, hypertension induces expression of platelet-derived growth factor,33 a known mitogen and survival factor for smooth muscle cells; therefore, it is possible that apoptosis follows withdrawal of tonic release of platelet-derived growth factor or other survival factors when wall tension is reduced. Finally, matrix constituents provide survival signals to many cells via integrin-mediated responses. For example, Boudreau et al34 showed that both ß1 integrin downregulation and extracellular matrix degradation can induce mammary epithelial cells to undergo apoptosis, as indicated by degradation of DNA into oligonucleosomes. Consequently, the massive upregulation of matrix-degrading enzymes observed in our studies may deprive cells of matrix-integrin interactions that are antiapoptotic. Conversely, cell-matrix interactions have been implicated in cell survival. For example, proteolytic degradation of collagen can release integrin ligands that interact with cellular integrins to convey survival signals to the cells.35
Although we believe that atrophy in the artery-cuffed artery is due to decreased circumferential wall tension, it is also possible that other consequences of cuffing contribute to medial atrophy, including obstruction of the transport of fluid and solutes across the media and disruption of the nutrient supply from adventitial vasa vasorum. However, previous studies appear to exclude these processes as causes of medial atrophy. Specifically, loosely fitting cuffs, which were sealed securely to the artery wall at both ends, obstructed transmedial fluid flux and adventitial blood supply but did not cause atrophy; instead, they elicited an intimal proliferative response.36 The primary differences between these cuffs and the tightly fitting cuffs we have employed is that the former do not constrain circumferential distension, but they are designed to totally obstruct transmedial fluid transport. Furthermore, the participation of medial tissue close to the lumen, ie, very close to the primary nutrient supply for wall tissue, argues against impaired nutrient delivery as a source of atrophy. We infer that off-loading tension is responsible for the atrophy that we observed. Available data on mechanical properties of rabbit carotid arteries37 indicate that wall tension was almost totally eliminated in our experiments; therefore, more modest off-loading may produce different or more subtle medial remodeling.
Application of the arterial cuff in this study also elicited an inflammatory response; however, this response was restricted to the suture line where the longitudinal cut in the cuffing artery was rejoined. Because medial atrophy was observed at all points around the circumference of the cuffed artery, we infer that this local inflammatory response did not contribute significantly to this response of the cuffed artery.
Finally, we were concerned that part of the remodeling response we observed may have been due to increased shear stress imposed by blood flow on the arterial wall. Increased shear stress was anticipated because the experimental procedure requires ligation of the contralateral carotid artery, and the remaining carotid artery therefore carries substantial collateral flow.14 15 Furthermore, the diameter reduction produced by the cuff was expected to increase shear stress. We found, however, that left external carotid artery ligation, which reduces left common carotid artery blood flow by 70%, had no effect on the remodeling response after cuffing. It appears, therefore, that the remodeling responses elicited by increased blood flow were totally masked by the very potent responses to application of the cuff.
|
Acknowledgments |
|---|
This work was supported by the Heart and Stroke Foundation of Ontario. B.L. Langille and S.L. Adamson are Career Investigators of the Heart and Stroke Foundation of Ontario. I.M. Bayer was supported by a studentship from the Department of Obstetrics and Gynecology, Mount Sinai Hospital.
Received September 3, 1998; accepted November 20, 1998.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
- Langille BL. Remodelling of developing and mature arteries: endothelium, smooth muscle and matrix. J Cardiovasc Pharmacol. 1993;21:S11–S17.
-
Langille BL, Bendeck MP, Keeley FW. Adaptations of
carotid arteries of young and mature rabbits to reduced carotid blood
flow. Am J Physiol. 1989;256:H931–H939.
[Abstract/Free Full Text] -
Kamiya A, Togawa T. Adaptive regulation of wall shear
stress to flow change in the canine carotid artery. Am J
Physiol. 1980;239:H14–H21.
[Abstract/Free Full Text] -
Heagerty AM, Aalkjær C, Bund SJ, Korsgaard N, Mulvany
MJ. Small artery structure in hypertension: dual processes of
remodeling and growth. Hypertension. 1993;21:391–397.
[Free Full Text] -
Davidson D. Pulmonary
hemodynamics at birth: effect of acute
cyclooxygenase inhibition in lambs. J
Appl Physiol. 1988;64:1676–1682.
[Abstract/Free Full Text] -
Thubrikar MJ, Baker JW, Nolan SP. Inhibition of
atherosclerosis associated with reduction of
arterial intraluminal stress in rabbits.
Arteriosclerosis. 1988;8:410–420.
[Abstract/Free Full Text] -
Courtman DW, Cho A, Langille BL, Wilson GJ.
Eliminating arterial pulsatile strain by external banding
induces medial but not neointimal atrophy and
apoptosis in the rabbit. Am J Pathol.. 1998;153:1723–1729.
[Abstract/Free Full Text] -
Cho A, Mitchell L, Koopmans D, Langille BL. Effects of
changes in blood flow rate on cell death and cell proliferation in
carotid arteries of immature rabbits. Circ Res. 1997;81:328–337.
[Abstract/Free Full Text] - LaBarca C, Paigen K. A simple, rapid and sensitive DNA assay procedure. Anal Biochem. 1980;102:344–352.[Medline] [Order article via Infotrieve]
-
Bendeck MP, Keeley FW, Langille BL. Perinatal
accumulation of arterial wall constituents: relation to
hemodynamic changes at birth. Am J
Physiol. 1994;267:H2268–H2279.
[Abstract/Free Full Text] - Kivirikko KI, Laitinen O, Prockop DJ. Modifications of a specific assay for hydroxyproline in urine. Anal Biochem. 1967;19:249–255.[Medline] [Order article via Infotrieve]
- Keeley FW, Morin JD, Vesely S. Characterization of collagen from normal human sclera. Exp Eye Res. 1984;39:535–542.
-
Cho A, Courtman DW, Langille BL. Apoptosis
(programmed cell death) in arteries of the neonatal lamb. Circ
Res. 1995;76:168–175.
[Abstract/Free Full Text] - Walpola PL, Gotlieb AI, Langille BL. Monocyte adhesion and changes in endothelial cell number, morphology, and F-actin distribution elicited by low shear stress in vivo. Am J Pathol. 1993;142:1392–1400.[Abstract]
- Di Stefano I, Koopmans DR, Langille BL. Modulation of arterial growth of the rabbit carotid artery associated with experimental elevation of blood flow. J Vasc Res. 1998;35:1–7.[Medline] [Order article via Infotrieve]
-
Bassiouny HS, Song RH, Hong XF, Singh A, Kocharyan H,
Glagov S. Flow regulation of 72-kD collagenase IV (MMP-2)
after experimental arterial injury. Circulation. 1998;98:157–163.
[Abstract/Free Full Text] -
Schwartz SM. Cell death and the caspase cascade.
Circulation. 1998;97:227–229.
[Free Full Text] -
DeBlois D, Tea B-S, Dam T-V, Tremblay J, Hamet P.
Smooth muscle apoptosis during vascular regression in
spontaneously hypertensive rats. Hypertension. 1997;29:340–349.
[Abstract/Free Full Text] - Hamet P, DeBlois D, Dam T-V, Richard L, Teiger E, Tea B-S, Orlov SN, Tremblay J. Apoptosis and vascular wall remodeling in hypertension. Can J Physiol Pharmacol. 1996;74:850–861.[Medline] [Order article via Infotrieve]
- Sharifi AM, Schiffrin EL. Apoptosis in aorta of deoxycorticosterone acetate-salt hypertensive rats: effect of endothelin receptor antagonism. J Hypertens. 1997;15:1441–1448.[Medline] [Order article via Infotrieve]
-
Nikkari ST, O'Brien KD, Ferguson M, Hatsukami T,
Welgus HG, Alpers CE, Clowes AW. Interstitial
collagenase (MMP-1) expression in human carotid
atherosclerosis. Circulation. 1995;92:1393–1398.
[Abstract/Free Full Text] - Li ZH, Li L, Zielke R, Cheng L, Xiao RP, Crow MT, Stetler-Stevenson G, Froehlich J, Lakatta EG. Increased expression of 72-kd type IV collagenase (MMP-2) in human aortic atherosclerotic lesions. Am J Pathol. 1996;148:121–128.[Abstract]
- Galis ZS, Sukhova GK, Libby P. Microscopic localization of active proteases by in situ zymography: detection of matrix metalloproteinase activity in vascular tissue. FASEB J. 1995;9:974–980.[Abstract]
- Nikkari ST, Geary RL, Hatsukami T, Ferguson M, Forough R, Alpers CE, Clowes AW. Expression of collagen, interstitial collagenase, and tissue inhibitor of metalloproteinases-1 in restenosis after carotid endarterectomy. Am J Pathol. 1996;148::777–783.
-
Leung DYM, Glagov S, Mathews MB. Elastin and collagen
accumulation in rabbit ascending aorta and pulmonary trunk
during postnatal growth: correlation of cellular synthetic response
with medial tension. Circ Res. 1977;41:316–323.
[Abstract/Free Full Text] - Zempo N, Kenagy RD, Au YPT, Bendeck M, Clowes MM, Reidy MA, Clowes AW. Matrix metalloproteinases of vascular wall cells are increased in balloon-injured rat carotid artery. J Vasc Surg. 1994;20:209–217.[Medline] [Order article via Infotrieve]
-
Bendeck MP, Irvin C, Reidy MA. Inhibition of matrix
metalloproteinase activity inhibits smooth muscle cell migration but
not neointimal thickening after arterial
injury. Circ Res. 1996;78:38–43.
[Abstract/Free Full Text] - Katsuda S, Okada Y, Imai K, Nakanishi I. Matrix metalloproteinase-9 (92-kd gelatinase/type IV collagenase equals gelatinase B) can degrade arterial elastin. Am J Pathol. 1994;145:1208–1218.[Abstract]
-
Isner JM, Kearney M, Bortman S, Passeri J.
Apoptosis in human atherosclerosis and
restenosis. Circulation. 1995;91:2703–2711.
[Abstract/Free Full Text] - Kockx M, De Meyer G. Apoptosis in human atherosclerosis and restenosis. Circulation. 1996;93:394–395.
- Han DKM, Haudenschild CC, Hong MK, Tinkle BT, Leon MB, Liau G. Evidence for apoptosis in human atherogenesis and in a rat vascular injury model. Am J Pathol. 1995;147:267–277.[Abstract]
- Bennett MR, Boyle JJ. Apoptosis of vascular smooth muscle cells in atherosclerosis. Atherosclerosis. 1998;138:3–9.[Medline] [Order article via Infotrieve]
- Negoro N, Kanayama Y, Haraguchi M, Umetani N, Nishimura M, Konishi Y, Iwai J, Okamura M, Inoue T, Takeda T. Blood pressure regulates platelet-derived growth factor A-chain gene expression in vascular smooth muscle cells in vivo: an autocrine mechanism promoting hypertensive vascular hypertrophy. J Clin Invest. 1995;95:1140–1150.
-
Boudreau N, Sympson CJ, Werb Z, Bissell MJ. Suppression
of ICE and apoptosis in mammary epithelial cells by
extracellular matrix. Science. 1995;267:891–893.
[Abstract/Free Full Text] -
Jones PL, Crack J, Rabinovitch M.
Regulation of tenascin-C, a vascular smooth muscle cell survival factor
that interacts with the
V ß3 integrin to promote epidermal
growth factor receptor phosphorylation and growth.
J Cell Biol. 1997;139:279–293.[Abstract/Free Full Text] -
De Meyer GRY, Van Put DJM, Kockx MM, Van Schil P,
Bosmans R, Bult H, Buyssens N, Vanmaele R, Herman AG. Possible
mechanisms of collar-induced intimal thickening. Arterioscler
Thromb Vasc Biol. 1997;17:1924–1930.
[Abstract/Free Full Text] - Cox RH. Comparison of carotid artery mechanics in the rat, rabbit, and dog. Am J Physiol. 1978;234:H280–H288
This article has been cited by other articles:
 |
|
 |
|
  S. Lehoux and A. Tedgui Making Up and Breaking Up: The Tortuous Ways of the Vascular Wall Arterioscler. Thromb. Vasc. Biol., May 1, 2005; 25(5): 892 - 894. [Full Text] [PDF]
|
|
|
|
 |
|
 S. Lehoux, C. A. Lemarie, B. Esposito, H. R. Lijnen, and A. Tedgui Pressure-Induced Matrix Metalloproteinase-9 Contributes to Early Hypertensive Remodeling Circulation, March 2, 2004; 109(8): 1041 - 1047. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E.-L. Marchand, S. Der Sarkissian, P. Hamet, and D. deBlois Caspase-Dependent Cell Death Mediates the Early Phase of Aortic Hypertrophy Regression in Losartan-Treated Spontaneously Hypertensive Rats Circ. Res., April 18, 2003; 92(7): 777 - 784. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I.-M. o Chung, H. K. Gold, S. M. Schwartz, Y. Ikari, M. A. Reidy, and T. N. Wight Enhanced extracellular matrix accumulation in restenosis of coronary arteries after stent deployment J. Am. Coll. Cardiol., December 18, 2002; 40(12): 2072 - 2081. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. S. Jackson, A. I. Gotlieb, and B. L. Langille Wall Tissue Remodeling Regulates Longitudinal Tension in Arteries Circ. Res., May 3, 2002; 90(8): 918 - 925. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. A. Fisher, B. L. Langille, and D. Srivastava Apoptosis During Cardiovascular Development Circ. Res., November 10, 2000; 87(10): 856 - 864. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Walsh, R. C. Smith, and H.-S. Kim Vascular Cell Apoptosis in Remodeling, Restenosis, and Plaque Rupture Circ. Res., August 4, 2000; 87(3): 184 - 188. [Full Text] [PDF]
|
|
|
|
 |
|
 B.-S. Tea, S. Der Sarkissian, R. M. Touyz, P. Hamet, and D. deBlois Proapoptotic and Growth-Inhibitory Role of Angiotensin II Type 2 Receptor in Vascular Smooth Muscle Cells of Spontaneously Hypertensive Rats In Vivo Hypertension, May 1, 2000; 35(5): 1069 - 1073. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Lemay, P. Hamet, and D. deBlois Losartan-induced apoptosis as a novel mechanism for the prevention of vascular lesion formation after injury Journal of Renin-Angiotensin-Aldosterone System, March 1, 2000; 1(1): 46 - 50. [Abstract] [PDF]
|
|
|
|
|
|
Z. S. Jackson, A. I. Gotlieb, and B. L. Langille Wall Tissue Remodeling Regulates Longitudinal Tension in Arteries Circ. Res., May 3, 2002; 90(8): 918 - 925. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||