© 1995 American Heart Association, Inc.
Articles |
Increased Elastin-Degrading Activity and Neointimal Formation in Porcine Aortic Organ Culture
Reduction of Both Features With a Serine Proteinase Inhibitor
From the Division of Cardiovascular Research (S.O., T.C., M.R.), Research Institute, The Hospital for Sick Children, and the Vascular Research Laboratory (E.W.Y.K., S.J.D., A.I.G.), The Toronto Hospital Research Institute and the Departments of Pediatrics, Pathology, and Medicine, University of Toronto, Canada.
Correspondence to Marlene Rabinovitch, MD, Division of Cardiovascular Research, The Hospital for Sick Children, 555 University Ave, Toronto, Ontario, Canada M5G 1X8.
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Abstract We investigated the association between tissue elastolytic activity and the development of neointimal formation using a previously described porcine aortic organ culture. Neointimal formation is associated with the presence of intact endothelium (nondenuded cultures) but is markedly reduced if endothelial cells are removed (denuded cultures). In nondenuded organ cultures, elastolytic activity assessed by using [3H]elastin increased sixfold at day 3 after initiation of the culture (P<.01), a time earlier than the previously published increase in intimal smooth muscle cells (ISMCs). Elastolytic activity did not increase from day 3 to day 7 despite doubling of ISMCs but did double by day 14 (P<.01) and remained elevated to day 28, correlating with increases in ISMCs. In denuded organ cultures, elastolytic activity was much lower than in nondenuded organ cultures at day 3 (P<.05) but increased fivefold in the presence of nondenuded organ culture conditioned medium (P<.01). Addition of
1-proteinase
inhibitor for 14 days caused a 60% decrease in elastolytic
activity in nondenuded organ cultures and a 27% reduction in ISMCs
compared with untreated controls (P<.05 for both). The
elastolytic activity, resolved as lytic bands on an elastin substrate
gel, reflected candidate enzymes, one at 76 kD and perhaps a doublet at
43 and 50 kD. Our study suggests that endothelial cells
release a soluble agent that enhances elastin-degrading activity in
the aorta and may at least partially account for the initiation of
neointimal formation.
Key Words: elastolytic activity • neointimal formation • aorta • 1-proteinase inhibitor • vascular disease
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Introduction |
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Numerous dynamic biological processes interact within the vessel wall to promote physiological and pathological conditions. Neointimal formation is a common feature of vascular disease, ie, atherosclerosis, vein graft stenosis, postangioplasty restenosis, postcardiac transplant coronary arteriopathy, and pulmonary vascular disease,1 2 but, despite extensive study, the pathophysiological mechanisms are incompletely understood. An aorta organ culture system has been developed as a useful model to investigate neointimal formation, since it allows the anatomic relationship of the vessel wall layers to be maintained and thus interactions between cells and matrix can be studied and understood. Studies from our group using a porcine aorta in organ culture in 5% FBS have shown that neointimal formation occurs in the presence of an intact, albeit injured, endothelium. However, neointimal formation is greatly reduced when endothelial cells are denuded, unless conditioned medium collected from nondenuded organ cultures is added. In the presence of an intact endothelium, the number of ISMCs appears to increase only after 3 days in culture; values are doubled by 7 days and continue to increase until day 14. Thereafter, the same number of ISMCs is observed up to 28 days in culture.3 4 5
Recent studies from our laboratory have shown increased pulmonary artery elastolytic activity in rats with pulmonary vascular disease induced by the toxin monocrotaline or by hypoxia. The enzymatic activity appears to be related to an endogenous vascular serine elastase, an enzyme of 20 kD related to the serine proteinase adipsin.6 Administration of elastase inhibitors appreciably reduced pulmonary hypertension and vascular changes7 8 including neointimal formation.7 We have also identified an enzyme similar in molecular weight and inhibitor profile associated with early features of graft arteriopathy in donor coronary arteries of piglets after heterotopic cardiac transplantation.9 It has also previously been shown in studies by Hornebeck et al10 11 that serine elastolytic activity in human aortic tissue increases with age and with atherosclerosis, suggesting a relation between elastolysis and the development of neointimal formation. More recently, in situ hybridization studies in atherosclerotic plaques have shown expression of a different enzyme, stromelysin, a matrix metalloproteinase with elastolytic activity.12
We therefore first determined whether there was elastolytic activity in
the porcine aortic organ culture prior to as well as coincident with
the times when an increase in the number of ISMCs was previously
reported.4 5 We characterized this enzymatic activity in
terms of its susceptibility to inhibitors. To address
further the nature of the relation between elastolytic activity and
neointimal formation, we measured elastolytic activity
under conditions in which neointimal formation is reduced.
The role of endothelium in the promotion of elastolytic
activity was studied by measuring elastolytic activity in denuded organ
cultures to which normal media or nondenuded organ culture media were
added. In the former, neointimal formation has been
observed to be reduced, while the addition of conditioned medium from
nondenuded cultures restores neointimal formation. Since we
found that elastolytic activity in the aorta was inhibited by
1-PI, we added this agent to the aortic organ culture
medium over a 14-day period and assessed whether this influenced the
development of neointimal formation. We further
characterized the elastolytic activity by zymography using elastin
substrate gel electrophoresis.
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Methods |
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Aortic Organ Culture
Porcine aortic organ cultures were prepared as previously described.3 Briefly, porcine thoracic aortas were obtained from a local slaughterhouse and transported in sterile phosphate-buffered saline and 2% antibiotics/antimycotics (GIBCO Laboratories). After removal of fat and adventitia, six rectangular pieces of full-thickness vessel wall (4x10 mm) were cut out with a scalpel blade, washed well with phosphate-buffered saline, and incubated in medium 199 (GIBCO) containing 5% FBS and 1% antibiotics/antimycotics. Aortas were maintained in culture for periods of 3, 7, 14, 21, and 28 days. The six segments from each aorta were pooled for the analyses, and comparisons were made using three or four different aortas studied at each time point. Complete removal of the adventitia and periadventitial fat has been verified by light-microscopic examination of the tissue.3 4 5
In other experiments, endothelial denudation was carried out by removing all surface endothelial cells using a single gentle stroke of a scalpel blade. This method has been previously confirmed to denude endothelium completely.4 Elastolytic activity of nondenuded aortic organ cultures was compared with that of denuded organ cultures. Comparisons were also made with denuded aorta organ cultures to which medium was added after collection from nondenuded organ cultures over a 24-hour period on day 4 of culture. This time point was chosen on the basis of previous studies in which it was shown that endothelial-conditioned media harvested on day 4 of culture would induce neointimal formation in denuded organ cultures by day 7.4 The elastolytic activity of the three different conditions of organ culture (nondenuded, denuded, and denuded plus 4-day conditioned medium) was compared after 3 days in culture. We analyzed tissue from four different aortas under each condition. Aortic organ cultures harvested at each time point were stored at -70°C.
Assay of Elastolytic Activity
The procedure for measuring elastolytic activity was described
by Hornebeck et al10 and modified by our
group.13 Briefly, the tissues were minced and then
homogenized in 0.9% saline using a Polytron (model PT
10/35, Kinematica). The homogenate was centrifuged
for 30 minutes at 4°C and the pellet was extracted twice for 18 hours
at 4°C in 1 mL of 0.5 mol/L Na acetate buffer, pH 4.0. The extracts
were pooled, dialyzed overnight against distilled water at 4°C, and
lyophilized. The lyophilized powder was reconstituted in 1 mL of 60%
saturated ammonium sulfate solution, shaken gently for 1 hour at room
temperature, placed at 4°C overnight to precipitate the proteins, and
then centrifuged for 1 hour at 4°C. All solutions contained 2
mmol/L methylamine to inhibit 2-macroglobulin. The
pellet was resuspended in 200 µL of 50 mmol/L Tris assay buffer, pH
8.0, containing 150 mmol/L NaCl, 10 mmol/L CaCl2,
0.02% polyoxyethylene 23 lauryl ether (Brij 35), and 0.02%
NaN3. Duplicate samples of 100 µL were incubated with 20
µL radiolabeled elastin substrate, and then an additional 100 µL of
Tris buffer was added to make up an end volume of 220 µL. The
radiolabeled elastin substrate was produced by labeling insoluble
bovine ligamentum nuchae elastin (Elastin Products Co) with
NaB3H4 (New England Nuclear) as described
previously.13 The specific activity of the
[3H]elastin was 2.0x103 cpm/µg protein.
The samples were incubated for 18 hours at 37°C, then
centrifuged for 4 minutes; 100 µL aliquots of supernatant
were added to 4 mL of ACS scintillation fluid, and the samples were
counted for 2 minutes by scintillation counter (1219 RackBeta, LKB
Wallac). In each assay, elastolytic activity was calculated by a
standard curve generated with HLE (Elastin Products Co, specific
activity 875 U/mg protein), and the mean value of the duplicate samples
was used in the calculation of elastolytic activity.
To characterize the elastolytic activity by its inhibitor
profile, nondenuded aorta organ cultures were prepared from two or
three different experiments. Duplicate samples were run and mean values
of percent inhibition over baseline were obtained using HLE as a
standard. The samples were preincubated with an inhibitor
for 30 minutes at 37°C before addition of [3H]elastin.
The inhibitors used were a metalloproteinase
inhibitor (2 mmol/L Na2EDTA) or serine
proteinase inhibitors [PMSF in a dose of 2 mmol/L, and
1-PI (25 and 50 µg/mL)] (Prolastin, Miles, Cutter
Biologicals and a gift kindly provided by Mary Ann Lark and Stan Beck).
The doses were chosen according to previously published
reports.7 14 15 16 That is, we16 and
others14 have shown that 2 mmol/L EDTA effectively
inhibits pseudomonas aeruginosa metalloelastase, and we have shown
that 2 mmol/L PMSF and 25 µg/mL 1-PI completely
inhibit pulmonary artery elastolytic activity.8
Because of incomplete inhibition of elastolytic activity in the aortic
organ culture at 25 µg/mL, we also used a higher dose of
1-PI (50 µg/mL). We also further characterized the
inhibitor profile of this elastolytic enzyme. We used 100
µmol/L E-64,17 a dose that will inhibit cysteine
proteinases.18 E-64 was kindly provided by Drs John Munger
and Harold Chapman, Harvard Medical School, Boston, Mass. We also used
a new elastase inhibitor, recombinant elafin, a gift of
Dr J-M Sallenave (McMaster University, Hamilton, Ontario, through
Zeneca-ICI, Macclesfield, England), in a dose of 3.8 µmol/L, which we
verified effectively inhibits both neutrophil
elastase19 and pulmonary artery elastase
(unpublished data).
To verify that the source of the elastin-degrading activity in the tissue was not due to tissue necrosis of the organ culture, we measured LDH in organ cultures on day 0 and day 4 (denuded and nondenuded). That is, we measured the amount released into the conditioned medium over the 24-hour period between day 0 and day 1 and day 4 and day 5. The 4-day time point was chosen since the increase in elastolytic activity was seen with nondenuded but not with denuded organ cultures. We measured LDH according to the method of Mitchell et al20 in which lactate dehydrogenase activity is measured spectrophotometrically as the conversion of NADH to NAD+ at 340 nm. To determine whether the [3H]elastin assay would detect other serine proteinases that have been implicated in neointimal formation,21 22 23 we added plasmin (2 µg/mL), thrombin (2 µg/mL), and trypsin (0.5% solution) and 10 to 100 U per mL of urokinase to the [3H]elastin without the addition of the tissue homogenate.
Experiments With Added 1-PI
In some experiments, we incubated aortas with added
1-PI at a concentration of 6 mg/mL added freshly every
other day. This dose was chosen after pilot experiments had shown that
with 3 mg/mL 1-PI there was no decrease in aortic
elastase activity after 2 weeks in organ culture. Tissues from nine
different aortas were examined at day 14, comparing intimal cell counts
from sections taken from each aorta incubated with and without the
inhibitor. The aorta sections were counted without
knowledge of the group to which they belonged, ie,
inhibitor treated or untreated. Tissues were fixed in 10%
buffered neutral formalin (BDH), dehydrated, cleared, and embedded in
paraffin. Sections 4 µm in thickness were stained with the
fluorescent nuclear dye Hoechst 33258 (Sigma Chemical Co), as
described previously.4 Nuclei were visualized under a
Zeiss photomicroscope III (x25 objective) equipped with a mercury
vapor lamp, epifluorescence optics, and interference
filters. The average number of ISMCs per field was calculated from 20
fields in each section. We further verified in these experiments that
the addition of 1-PI did not alter
endothelial cell morphology or function as judged by
uptake of acetylated LDL.4
Elastin Substrate Gel Electrophoresis
Aortic tissue taken after 14 days in organ culture was extracted
as described above for the elastase activity assay except that the
pellets were diluted into sample buffer (0.4 mol/L Tris, pH 6.8, 5%
SDS, 20% glycerol, 0.03% bromophenol blue) and loaded onto a 12% SDS
polyacrylamide gel which contained kappa-elastin (1 mg/mL)
as previously reported.6 Electrophoresis was carried out
at 100 V until the dye front reached the bottom. The gel was removed
and incubated for 1 hour at room temperature in 100 mL of 2.5% Triton
X-100 on a rotary shaker. The Triton X-100 solution was then replaced
with 100 mL of Tris assay buffer (50 mmol/L Tris, pH 8.0, 150 mmol/L
NaCl, 10 mmol/L CaCl2, 0.02% Brij-35), and
incubation was continued at 37°C for 4 days. The gel was then stained
with Coomassie blue and destained. Areas of elastase activity are
visualized as nonstaining regions of the gel.
Statistical Analysis
The values were expressed as mean±SE. All statistical
analyses were performed using either a Student's t
test when only two groups were compared or, when more than two groups
were compared, a one-way ANOVA followed by Tukey's test of
multiple comparisons to establish which groups were different.
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Results |
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Elastolytic Activity of Nondenuded Organ Cultures
Elastolytic activity was increased in nondenuded organ cultures by sixfold as early as day 3, values were similar between days 3 and 7, and then increased a further twofold at day 14. Thereafter, elastolytic activity remained elevated up to day 28 (Fig 1
). The
rise in elastolytic activity thus preceded the increase in ISMCs that
was observed only after day 34 and it is of further
interest that there was no change in elastolytic activity between days
3 and 7 despite the fact that the number of ISMCs doubled over this
time period.4 The further doubling in elastolytic activity
between days 7 and 14 is correlated with a further doubling in intimal
cell number over this time period.4 The sustained increase
in elastolytic activity between days 14 and 28 correlates with a
sustained increase in the number of ISMCs.4
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Elastolytic Activity of Denuded Organ Cultures
Elastolytic activity of denuded organ cultures at day 3 was about
one sixth that of nondenuded organ cultures and, in fact, values were
similar to those observed in nondenuded organ cultures at day 0. When
incubated in conditioned medium collected from nondenuded aortic organ
cultures, elastolytic activity of denuded organ cultures increased
fivefold and values approached those of nondenuded cultures (Fig 2). This was after only 3 days, at a time point prior to
the previously observed increase in the number of ISMCs, which occurred
only after a 7-day incubation in nondenuded cultured conditioned
media.4 There was no detectable elastolytic activity in
the conditioned media, suggesting that an endothelial
factor was in fact inducing the increase in the aorta tissue.
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The elastolytic activity could not be attributed to tissue necrosis of the organ culture since there was no difference in LDH activity measured in the culture media when day 0 cultures were compared with day 4 cultures, denuded or nondenuded (values in IU per L=13.1±3.5 SEM, 14.5±1.4 SEM, and 14.0±2.6 SEM, respectively) (P>.05).
The inhibitor profile of the nondenuded aorta organ culture
elastolytic activity assayed from tissue obtained from days 4 to 14
(Fig 3) was different from that of the pulmonary
artery elastase we had previously described.6 It was
compatible with that of a serine proteinase in that the elastolytic
activity was 60% inhibited by low-dose (25 µg/mL) and 80%
inhibited by high-dose (50 µg/mL) 1-PI with no
inhibition evident with Na2EDTA at a dose that would
inhibit metalloelastases16 or with E-64 at a dose that
would inhibit cysteine proteinases.17 18 The
inhibitor profile was unusual in that there was only
incomplete (30%) inhibition with a nonspecific serine PI such as PMSF
and no inhibition with the more specific neutrophil elastase
inhibitor elafin.19 We were unable to show
that other serine proteinases such as urokinase would degrade elastin
as judged by cpm above baseline. We did, however, identify some
degradation of the substrate in the range observed in the tissue but
with high-dose commercial preparations of plasmin, thrombin, and
trypsin. That is, it required 2 µg of thrombin or plasmin or 0.5%
trypsin to degrade the amount of elastin equivalent to 4 ng of HLE.
This is the equivalent concentration of the elastolytic activity
measured in our assays. These concentrations of serine proteinases
would be unlikely in the amount of tissue assayed. Moreover, they would
be inhibited by PMSF.
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1-PI, Elastolytic Activity, and ISMC
Counts
The elastolytic activity in the tissue after a 14-day incubation
with 6 mg/mL 1-PI was reduced by 60% over values in
tissues incubated in the absence of the inhibitor (Fig 4). The number of ISMCs in nondenuded aortic organ
cultures at day 14 (56.31±4.14) was reduced by 27% when cultures were
treated with 1-PI (40.93±3.92, P<.05) (Fig 5). Fig 6 shows
representative photomicrographs. We confirmed that
there was no adverse effect of 1-PI on
endothelial cell function or morphology as judged by
uptake of acetylated LDL (photomicrographs not shown).
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Elastin Substrate Gel Electrophoresis
The elastin substrate gel from the 14-day aortic organ culture
extract revealed a lytic band at approximately 76 kD, and there
appeared to be a lytic doublet at 43 and 50 kD (Fig 7).
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Discussion |
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In this study on elastin degrading proteinases, we have shown that elastin-degrading activity of porcine aortic organ cultures increased by sixfold at day 3, preceding the increase in ISMCs previously published.4 5 There was no further increase in elastolytic activity between days 3 and 7 despite the doubling in cell number previously reported.4 5 This indicates that the initial increase in elastin-degrading activity was not merely a function of the increase in the number of cells in the aortic organ culture. The increase in elastolytic activity observed between days 7 and 14, however, does in fact correlate closely with a comparable increase in the number of ISMCs. While we normalized all data per mg tissue, we cannot exclude the possibility that the increase in enzymatic activity over the 7- to 14-day time period may have been a function of the increase in the number of ISMCs. The elastolytic activity was resolved on an elastin substrate gel as lytic bands, one at 76 kD and what appeared to be a doublet at 43 and 50 kD. This feature in addition to the inhibitor profile distinguishes the elastolytic enzyme(s) in the aortic organ culture tissue from vascular serine elastases described by our group6 9 and others.11
The increase in elastolytic activity in organ culture, much like the intimal proliferative response, proved to be endothelium-dependent, since elastolytic activity of denuded aortas did not increase in culture but was similar to that of nondenuded aortas when cultured in conditioned medium from those vessels. The increase in elastolytic activity was observed at a time point after incubation with the culture medium (3 days), which preceded the increase in neointimal SMC number. We verified that the conditioned medium did not contain elastolytic activity, so it appears that an endothelial factor may activate or induce the synthesis of an elastase produced by nondenuded organ cultures. It remains to be proved whether this is the same factor necessary for the neointimal formation that has been observed when denuded cultures are incubated with conditioned medium. Although elastases are produced by neutrophils,24 macrophages,25 monocytes,26 endothelial cells,27 and platelets,28 as well as SMCs,9 13 29 the denuded organ cultures in which elastase activity was measured contain only SMCs.4 5
We can speculate that an early increase in elastolytic activity may
induce ISMC proliferation in this aorta organ culture system through
the liberation or activation of growth factors.30 31 32 33 34 35
Proteinases can release or activate growth factors, such as
basic fibroblast growth factor30 31 and transforming
growth factor-ß,32 33 which are stored in the
extracellular matrix. In addition, proteinases with elastolytic
activity can also process growth factors, such as transforming growth
factor-, at cell surfaces.35 36 An interesting
study37 recently showed that coculture of leukocytes with
endothelial cells caused mitogenic activity
that could be reproduced by elastolytic enzymes.
The mechanism for the neointimal proliferation observed in aortic organ culture has been attributed to perturbation of the endothelium as judged by high turnover.4 The features of this perturbation have been largely unexplored, eg, the relation to expression of adhesion molecules such as intercellular adhesion molecules-1 and -2 and vascular cell adhesion molecule-1.38 39 40 These molecules are expressed in response to cytokines,41 and signaling through these adhesion molecules can induce the release of proteolytic enzymes from other cells.42 Unfortunately, it was not feasible to investigate expression of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 in our system since antibodies that recognize these adhesion molecules do not cross-react with porcine species. While it is possible that the "activated" state of the endothelium does in fact signal the release of an elastase-inducing factor, it could also be that SMCs are perturbed or activated in some way that facilitates release of the elastase by the endothelial factor or proliferation in response to elastase activity. We have shown in other studies that a serum factor can induce release of a serine elastase29 from cultured pulmonary or systemic SMCs at least as judged by its inhibitor profile, and our unpublished data have shown that cultured endothelial cells can release a similar factor.
The neointimal proliferation in the porcine aortic organ culture is largely attributed to proliferation of resident ISMCs with migration of SMCs being of much less importance.4 This differs from other models of neointimal formation in which ISMCs are not present normally. For example, in the rat carotid balloon injury model, neointimal proliferation is associated with PDGF-related SMC migration, with activation of the serine proteinase plasminogen activator21 22 23 and more recently, metalloproteinases.43 44 Nonelastolytic metalloproteinases are also seen in atherosclerotic plaques45 and associated with migrating SMCs.46
Elastolytic activity may be only one of the proteolytic properties of
the vascular enzyme we have identified in aorta organ culture, since
other elastases, such as neutrophil elastase, have been shown
to degrade fibronectin,47 laminin,48 type IV
collagen,49 and proteoglycans,50 and
alterations in extracellular matrix proteins may also influence cell
migration51 52 and proliferation.53 The
elastin-degrading activity of aortic organ cultures was inhibited
by 1-PI but not by Na2EDTA or E-64,
suggesting that this enzyme is a serine proteinase. This
inhibitor profile is different from that previously
described in pig coronary artery tissue9 and
SMCs13 29 and in rat pulmonary
artery6 7 13 since there is incomplete inhibition with
PMSF, a nonspecific serine proteinase inhibitor, and there
is no inhibition with elafin, a more specific elastase
inhibitor. The elastolytic activity observed is not likely
related to other serine proteinases such as urokinase implicated in
cell proliferation or plasminogen activator
associated with cell migration. We were unable to show degradation of
the elastin substrate with urokinase, and the concentrations of plasmin
required to degrade the equivalent amount of elastin in this assay
would be unexpected in aortic tissue (ie, in the microgram as opposed
to the nanogram range). Moreover, both these serine proteinases would
be completely inhibited by PMSF. We were, however, able to show only
weak inhibition of elastolytic activity with PMSF.
The inhibitor profile is, however, unusual, and the lack of
complete inhibition with PMSF or low-dose 1-PI as
well as the lack of inhibition with elafin may reflect relative
inaccessibility of these inhibitors, given the amount of
elastin in the tissue homogenates available for binding to
the enzyme. It is also possible that this is a serine proteinase with
only weak elastolytic activity. There was no evidence of tissue
necrosis that would explain the elastolytic activity in nondenuded
organ cultures as judged by LDH levels. Moreover, it would be unlikely
that the 4-day endothelial-denuded organ cultures
would have less necrosis than the nondenuded cultures. Our experiments
required a very high dose (6 mg/mL) of 1-PI to inhibit
the elastin-degrading activity of the organ culture tissue,
especially in comparison with the dose (50 µg/mL) used to get 80%
inhibition in the tissue homogenate. This is perhaps
because tremendous excess of the inhibitor was necessary to
penetrate into the tissue.
Our recent study in rats with monocrotaline-induced
pulmonary hypertension has shown that pulmonary artery
elastolytic activity increased at 2 days after injection of the toxin
and that this increase precedes fragmentation of the internal elastic
lamina, which is observed at day 4, and intimal proliferation, which is
seen much later at day 21.7 Fragmentation of elastin has
also been observed in the early stages of
atherosclerosis.54 We have shown that
administration of serine PIs, such as SC-37698 (Searle) and
1-PI, to rats injected with monocrotaline or exposed to
hypoxia reduced intimal proliferation8 as well as
pulmonary hypertension.7 8 We therefore reasoned
that long-term administration of these inhibitors could
indicate a possible cause-and-effect relationship between the
elevation in elastolytic activity and the development of
neointimal formation. It was therefore interesting that
long-term administration of 1-PI did, in fact,
reduce both elastolytic activity in the aortic organ culture tissue and
the extent of neointimal formation, as judged by the number
of ISMCs.
We cannot exclude the possibility that the effect of
1-PI in reducing the smooth muscle proliferative
response associated with neointimal formation in aortic
organ culture may have been related to a reduction in the activity of
other nonelastolytic serine proteinases or that it may have been
unrelated to proteinase inhibition. Clowes et al21
reported the activity of tissue-type plasminogen
activator to be increased in rat carotid artery after
balloon catheter injury, which was related to SMC migration and
expression of PDGF. Observations made by Jackson and
Reidy,23 however, suggested that different serine
proteinase inhibitors could decrease neointimal
formation by effects that were related as well as unrelated to their
reduction in plasminogen activator
activity.
Elastolytic activity of organ cultures increased up to day 14. Thus, despite the elevated levels of enzymatic activity, there was no further increase in numbers of ISMCs. It is not known why, as previously observed, neointimal formation plateaus after 21 days, but it is possible that increased production or release of growth inhibitors, such as heparin55 and heparan sulfate,56 may be responsible and may counteract the effects of elevated elastase activity.
In conclusion, we have shown that increased elastolytic activity was
associated with neointimal formation in porcine aortic
organ culture and that the endothelium appears to
produce a factor that increases elastolytic activity in the aortic
organ culture. Further studies are required to understand how this
increased elastolytic activity may play a mechanistically important
role in neointimal formation in this model. The usefulness
of 1-PI in reducing the ISMC proliferative response may
offer a new approach to regulating vascular lesions arising due to SMC
proliferation.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This work was supported by grants T-2229, B-1964, and T-2351 from the Heart and Stroke Foundation of Ontario. S.O. was supported in part by a scholarship from the University of Tokyo, E.W.Y.K. and S.J.D. by studentships, T.C. by a Medical Scientist Award of the Heart and Stroke Foundation of Canada, and M.R. by a Clinical Investigator Award of the Heart and Stroke Foundation of Ontario. We gratefully acknowledge the excellent secretarial assistance of Joan Jowlabar.
Received May 3, 1994; accepted September 8, 1995.
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References |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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1. Ip JH, Fuster V, Badimon L, Badimon J, Taubman MB, Chesebro JH. Syndromes of accelerated atherosclerosis: role of vascular injury and smooth muscle cell proliferation. J Am Coll Cardiol. 1990;15:1667-1687. [Abstract]
2. Heath D, Edwards J. The pathology of hypertensive pulmonary vascular disease. Circulation. 1958;18:533-547. [Medline] [Order article via Infotrieve]
3. Gotlieb AI, Boden P. Porcine aortic organ culture: a model to study the cellular response to vascular injury. In Vitro. 1984;20:535-542. [Medline] [Order article via Infotrieve]
4. Koo EWY, Gotlieb AI. Endothelial stimulation of intimal cell proliferation in a porcine aortic organ culture. Am J Pathol. 1989;134:497-503. [Abstract]
5. Koo EWY, Gotlieb AI. Neointimal formation in the porcine aortic organ culture, I: cellular dynamics over 1 month. Lab Invest. 1991;64:743-753. [Medline] [Order article via Infotrieve]
6. Zhu L, Wigle D, Hinek A, Kobayashi J, Ye C, Zuker M, Dodo H, Keeley FW, Rabinovitch M. The endogenous vascular elastase that governs development and progression of monocrotaline-induced pulmonary hypertension in rats is a novel enzyme related to the serine proteinase adipsin. J Clin Invest. 1994;94:1163-1171.
7.
Ye C, Rabinovitch M. Inhibition of
elastolysis by SC-37698 reduces development and progression of
monocrotaline pulmonary hypertension. Am J
Physiol. 1991;261:H1255-H1267.
8. Maruyama K, Ye C, Woo M, Venkatacharya H, Lines LD, Silver MM, Rabinovitch M. Chronic hypoxic pulmonary hypertension in rats and increased elastolytic activity. Am J Physiol. 1991;261:1716-1726.
9. Oho S, Rabinovitch M. Post-cardiac transplant arteriopathy in piglets is associated with fragmentation of elastin and increased activity of a serine elastase. Am J Pathol. 1994;145:202-210. [Abstract]
10. Hornebeck W, Adnett JJ, Robert L. Age dependent variation of elastin and elastase in aorta and human breast cancers. Exp Gerontol. 1978;13:293-298. [Medline] [Order article via Infotrieve]
11. Hornebeck W, Derouette JC, Robert L. Isolation, purification and properties of aortic elastase. FEBS Lett. 1975;58:66-70. [Medline] [Order article via Infotrieve]
12.
Henney AM, Wakeley PR, Davies MJ, Foster K, Hembry R,
Murphy G, Humphries S. Localization of stromelysin gene
expression in atherosclerotic plaques by in situ hybridization.
Proc Natl Acad Sci U S A. 1991;88:8154-8158.
13.
LaBourene JI, Coles JG, Johnson DJ, Mehra A, Keeley FW,
Rabinovitch M. Alterations in elastin and collagen
related to the mechanism of progressive pulmonary venous
obstruction in a piglet model: a hemodynamic,
ultrastructural and biochemical study. Circ
Res. 1990;66:438-456.
14. Banda MJ, Werb Z. Mouse macrophage elastase: purification and characterization as a metalloproteinase. Biochem J. 1981;193:589-605. [Medline] [Order article via Infotrieve]
15. Leake DS, Hornebeck W, Brechemier D, Robert L, Peters TJ. Properties and subcellular localization of elastase-like activities of arterial smooth muscle cells in culture. Biochem Biophys Acta. 1983;761:41-47. [Medline] [Order article via Infotrieve]
16. Todorovich-Hunter L, Dodo H, Ye C, McCready L, Keeley FW, Rabinovitch M. Increased pulmonary artery elastolytic activity in adult rats with monocrotaline-induced progressive hypertensive pulmonary vascular disease compared with infant rats with nonprogressive disease. Am Rev Respir Dis. 1992;146:213-223. [Medline] [Order article via Infotrieve]
17. Barrett AJ, Kembhavi AA, Brown MA, Kirschke H, Knight CG, Tamai M, Hanada K. L-trans-Epoxysuccinyl-leucylamido (4-guanidino)butane (E-64) and its analogs as inhibitors of cysteine proteinases including cathepsins B, H and L. Biochemistry. 1984;201:189-198.
18.
Shi GP, Munger JS, Meara JP, Rich DH, Chapman HA.
Molecular cloning and expression of human alveolar
macrophage cathepsin S, an elastinolytic cysteine
protease. J Biol Chem. 1992;267:7258-7262.
19. Sallenave JM, Ryle AP. Purification and characterization of elastase-specific inhibitor: sequence homology with mucus proteinase inhibitor. Biol Chem Hoppe Seyler. 1991;372:13-21. [Medline] [Order article via Infotrieve]
20. Mitchell DB, Santone KS, Acosta D. Evaluation of cytotoxicity in cultured cells by enzyme leakage. J Tissue Culture Methods. 1980;6:113-116.
21.
Clowes AW, Clowes MM, Au YPT, Reidy MA, Belin D.
Smooth muscle cells express urokinase during mitogenesis and
tissue-type plasminogen activator during
migration in injured rat carotid artery. Circ
Res. 1990;67:61-67.
22.
Jackson CL, Raines EW, Ross R, Reidy MA. Role of
endogenous platelet-derived growth factor in
arterial smooth muscle cell migration after balloon
catheter injury. Arterioscler Thromb. 1993;13:1218-1226.
23. Jackson CL, Reidy MA. The role of plasminogen activation in smooth muscle cell migration after arterial injury. Ann N Y Acad Sci. 1992;667:141-150. [Medline] [Order article via Infotrieve]
24. Janoff A, Scherer J. Mediators of inflammation in leukocyte lysosomes, IX: elastinolytic activity in granules of human polymorphonuclear leukocytes. J Exp Med. 1968;128:1137-1155. [Abstract]
25.
Werb Z, Gordon S. Elastase secretion by
stimulated macrophages: characterization and
regulation. J Exp Med. 1975;142:361-377.
26. Lavie G, Zucker-Franklin D, Franklin EC. Elastase-type proteases on the surface of human blood monocytes: possible role in amyloid formation. J Immunol. 1980;125:175-180. [Abstract]
27. Laug WE, Weinblatt ME, Jones PA. Endothelial cells degrade extracellular matrix proteins produced in vitro. Thromb Haemost. 1985;54:498-502. [Medline] [Order article via Infotrieve]
28. Robert B, Szigeti M, Robert L, Legrand Y, Pignaud G, Caen J. Release of elastolytic activity from blood platelets. Nature. 1970;227:1248-1249. [Medline] [Order article via Infotrieve]
29. Kobayashi J, Wigle D, Childs T, Zhu J, Keeley FW, Rabinovitch M. Serum-induced vascular smooth muscle cell elastolytic activity through tyrosine kinase intracellular signalling. J Cell Physiol. 1994;160:121-131. [Medline] [Order article via Infotrieve]
30.
Saksela O, Moscatelli D, Sommer A, Rifkin DB.
Endothelial cell-derived heparin sulfate
binds basic fibroblast growth factor and protects it from proteolytic
degradation. J Cell Biol. 1988;107:743-751.
31.
Klagsbrun M, Edelman ER. Biological and
biochemical properties of fibroblast growth factors: implications for
the pathogenesis of atherosclerosis.
Arteriosclerosis. 1989;9:269-278.
32.
Lyons RM, Keski-Oja J, Moses HL. Proteolytic
activation of latent transforming growth factor beta from fibroblast
conditioned medium. J Cell Biol. 1988;106:1659-1665.
33.
Sato Y, Rifkin DB. Inhibition of
endothelial cell movement by pericytes and smooth
muscle cells: activation of a latent form of transforming growth factor
beta 1-like molecule by plasmin during co-culture.
J Cell Biol. 1989;109:309-315.
34.
Taipale J, Koli K, Keski-Oja J. Release of
transforming growth factor ß1 from the pericellular matrix of
cultured fibroblasts and fibrosarcoma cells. J
Biol Chem. 1992;267:25378-25384.
35.
Mueller SG, Patterson AJ, Kudlow JE.
Transforming growth factor alpha in arterioles: cell surface
processing of its precursor by elastase. Mol Cell
Biol. 1990;10:4596-4602.
36.
Cappelluti E, Strom SC, Harris RB. Potential
role of two novel elastase-like enzymes in processing
pro-transforming growth factor-.
Biochemistry. 1993;32:551-560. [Medline]
[Order article via Infotrieve]
37.
Totani L, Piccoli A, Pelligrini G, DiSanto A, Lorenzet
R. Polymorphonuclear leukocytes enhance release of growth
factors by cultured endothelial cells.
Arterioscler Thromb. 1994;14:125-132.
38. Albelda SM, Smith CW, Ward PA. Adhesion molecules and inflammatory injury. FASEB J. 1994;8:504-512. [Abstract]
39. Li H, Cybulsky M, Gimbrone M, Libby P. An atherogenic diet rapidly induces VCAM-1, a cytokine-regulatable mononuclear leukocyte adhesion molecule, in rabbit aortic endothelium. Arterioscler Thromb. 1993;143:197-204.
40.
Osborn L, Vassallo C, Benjamin C.
Activated endothelium binds lymphocytes
through a novel binding site in the alternatively spliced domain of
vascular cell adhesion molecule-1. J Exp
Med. 1992;176:99-107.
41. Briscoe D, Cotran R, Pober J. Effects of tumor necrosis factor, lipopolysaccharide and IL-4 on the expression of vascular cell adhesion molecule-1 in vivo. J Immunol. 1992;149:2954-2960. [Abstract]
42.
Romanic A, Madri J. The induction of 72kD
gelatinase in T cells upon adhesion to endothelial
cells is VCAM-1 dependent. J Cell Biol. 1994;125:1165-1178.
43. Zempo N, Kenagy RD, Au YPT, Clowes MM, Clowes AW. A smooth muscle metalloproteinase is increased in injured rat carotid artery. FASEB J. 1993;7:A269. Abstract.
44. Bendeck MP, Reidy MA. Matrix metalloproteinase expression after carotid arterial injury in the rat. J Cell Biochem. 1994;295:S18A. Abstract.
45. Sukhova G, Galis Z, Lark MW, Lee RT, Libby P. Regional expression of matrix-degrading proteinases in human atherosclerotic plaques. Circulation. 1993;88:I-174. Abstract.
46. Pauly RR, Monticone R, Lakatta EG, Crow MT. The migration of vascular smooth muscle cells through a reconstituted basement membrane barrier requires 72kD type IV gelatinase and is suppressed by differentiation. Circulation. 1993;88:I-174. Abstract.
47.
McDonald JA, Kelley DG. Degradation of
fibronectin by human leukocyte elastase: release of biologically
active fragments. J Biol Chem. 1980;255:8848-8858.
48. Heck LW, Blackburn WD, Irwin MH, Abrahamson DR. Degradation of basement membrane laminin by human neutrophil elastase and cathepsin G. Am J Pathol. 1990;136:1267-1274. [Abstract]
49.
Mainardi CL, Dixit SN, Kang AH. Degradation of
type IV (basement membrane) collagen by a proteinase isolated from
human polymorphonuclear leukocyte granules. J
Biol Chem. 1980;255:5435-5441.
50. Roughley PJ, Barrett AJ. The degradation of cartilage proteoglycans by tissue proteinases: proteoglycan structure and its susceptibility to proteolysis. Biochem J. 1977;167:629-637. [Medline] [Order article via Infotrieve]
51. Boudreau N, Turley E, Rabinovitch M. Fibronectin, hyaluronan and a hyaluronan binding protein contribute to increased ductus arteriosus smooth muscle cell migration. Dev Biol. 1991;143:235-247. [Medline] [Order article via Infotrieve]
52.
Werb Z, Tremble PM, Behrendtsen O, Crowley E, Damsky
CH. Signal transduction through the fibronectin receptor induces
collagenase and stromelysin gene expression.
J Cell Biol. 1989;109:877-889.
53.
Majack RA, Cook SC, Bornstein P. Control of
smooth muscle cell growth by components of the extracellular matrix:
autocrine role for thrombospondin. Proc Natl Acad Sci
U S A. 1986;83:9050-9054.
54.
Ross R, Glomset JA.
Arteriosclerosis and the
arterial smooth muscle cell. Science. 1973;180:1332-1339.
55.
Castellot JJ Jr, Favreau LV, Karnovsky MJ, Rosenberg
RD. Inhibition of vascular smooth muscle cell growth by
endothelial cell derived heparin: possible role of a
platelet endoglycosidase. J Biol Chem. 1982;257:11256-11260.
56. Benitz WE, Kelley RT, Anderson CM, Lorant DE, Bernfield M. Endothelial heparan sulfate proteoglycan, I: inhibitory effects on smooth muscle cell proliferation. Am J Respir Cell Mol Biol. 1990;2:13-24.
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