1 Department of Physiology and Biophysics and 2 Department of Cardiothoracic Surgery, School of Medicine, The University of Mississippi Medical Center, Jackson, Mississippi 39216
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ABSTRACT |
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To test the hypothesis that
homocysteine induces constrictive vascular remodeling by inactivating
peroxisome proliferator-activated receptor (PPAR), aortic endothelial
cells (ECs) and smooth muscle cells (SMCs) were isolated. Collagen gels
were prepared, and ECs or SMCs (105) or SMCs + ECs
(104) were incorporated into the gels. To characterize
PPAR, agonists of PPAR- [ciprofibrate (CF)] and PPAR-
[15-deoxy-12,14-prostaglandin J2 (PGJ2)] were
used. To determine the role of disintegrin metalloproteinase (DMP),
cardiac inhibitor of metalloproteinase (CIMP) was used in collagen
gels. Gel diameter at 0 h was 14.1 ± 0.2 mm and was unchanged up to 24 h as measured by a digital micrometer. SMCs reduce gel diameter to 10.5 ± 0.4 mm at 24 h. Addition of
homocysteine to SMCs reduces further the gel diameter to 8.0 ± 0.2 mm, suggesting that SMCs induce contraction and that the
contraction is further enhanced by homocysteine. Addition of ECs and
SMCs reduces gel diameter to 12.0 ± 0.3 mm, suggesting that ECs
play a role in collagen contraction. Only PGJ2, not
CF, inhibits SMC contraction. However, both PGJ2 and CF
inhibit contraction of ECs and SMCs + ECs. Addition of anti-DMP
blocks SMC- as well as homocysteine-mediated contraction. However, CIMP
inhibits only homocysteine-mediated contraction. The results suggest
that homocysteine may enhance vascular constrictive remodeling by
inactivating PPAR-
and -
in ECs and PPAR-
in SMCs.
aorta; arteriosclerosis; hypertension; peroxisome proliferator-activated receptor; fibrate; prostaglandin; endothelial cell; smooth muscle cell
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INTRODUCTION |
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ARTERIAL WALL
REMODELING is one of the most important factors regulating lumen
diameter after acute and/or chronic vascular injury (7, 28, 29,
44). Smooth muscle cells (SMCs) remodel the existing and
new extracellular matrix (ECM). In response to ECM degradation, SMCs
alter phenotype (43). The consequences of remodeling may
lead to alterations in arterial wall geometry and lumen diameter
(7, 28, 29, 44). Although the extracellular environment
strongly influences cell behavior, it is unclear whether the changes in
matrix composition affect connective tissue shrinkage. Hyperhomocysteinemia is associated with hypertension (39)
and increases vascular intimal-medial thickness (26, 34).
Homocysteine causes arteriosclerosis (19, 36, 40),
endothelial cell desquamation (38), thromboresistance
(22), SMC proliferation (41, 45), collagen
synthesis (23, 45), oxidation of low-density lipoprotein (12), increased monocyte adhesion to the vessel wall
(20), platelet aggregation (6), coagulation
(34), blood rheology (8, 25), and activation
of plasminogen and metalloproteinase (18, 47), the two
neutral proteinases associated with remodeling. Previous studies from
our laboratory have identified a redox-sensitive homocysteine receptor
in SMC. This receptor regulates collagen expression (45).
Primarily, there are two nuclear transcription factor (NF) receptors
that control the redox state of the cell. NF-B is induced by
homocysteine (3, 49). Peroxisome proliferator-activated receptor (PPAR) is proantioxidant. In addition, a negative correlation between high levels of homocysteine and PPAR expression has been demonstrated (4, 14). PPAR, upon induction, promotes the synthesis of superoxide dismutase (SOD) and catalase (16,
33). Meanwhile, PPAR decreases NADH/NADPH oxidase (15,
16). The high levels of homocysteine are associated with
increased oxyradical generation (1) and oxidative injury
(30, 52). The agonists of PPAR decrease the oxidative
stress and metalloproteinase activity in macrophages (24,
35), decrease the mRNA of plasminogen activator and increase the
mRNA of plasminogen activator inhibitor (50), and decrease
the intimal-medial thickness (27). It is unclear, however,
whether the induction of PPAR regresses intimal-medial thickness
by decreasing homocysteine-mediated metalloproteinase activation. The hypothesis is that homocysteine induces constrictive collagen remodeling by antagonizing PPAR and increasing disintegrin metalloproteinase (DMP) activity.
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MATERIALS AND METHODS |
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Cell cultures.
A segment of human aorta was obtained at the time of cardiac transplant
from patients with idiopathic dilated cardiomyopathy who had apparently
normal vessels. An Institutional Review Board waiver was obtained
before the tissue was collected. The aorta was opened longitudinally,
placed in Dubecco's minimal essential medium (DMEM), and used within
30 min. Endothelial cells (ECs) were removed by gently scraping the
lumen with a cotton swab. The swab was immersed in DMEM
containing 10% fetal calf serum (FCS). The SMCs were isolated from
medial tissue after the adventitia had been carefully separated and the
tissue had been minced with collagenase (20 µg/mg of tissue) in DMEM
at 37°C for 2 h. The cells were washed two times with DMEM
before they were plated onto 6-cm culture dishes. The cells were
cultured in DMEM, 10% FCS, 100 IU/ml penicillin/streptomycin at 37°C
and 5% CO2. The cells were passaged when nearly confluent
and studied between passages 3 and 6.
The ECs were characterized by their cobblestone appearance and positive
staining for von Willebrand factor VIII (46). The SMCs
were identified by their spindle shape and positive staining for smooth
muscle -actin (46).
Collagen gel contraction assay. Collagen gels were prepared as described (43). Briefly, 24-well plates were precoated with 1% agarose to promote gel detachment (48). Type I collagen (Southern Biochemical) was diluted with 4× DMEM and cell suspensions so that the final mixture resulted in 1.25 mg/ml collagen. Gelation occurred within 10 min at 37°C. The confluent cells, serum-deprived for 24 h, were suspended in collagen gel suspension. DMEM containing 0.2% FCS was added to each well. The gels were lifted off the bottom of the wells and allowed to float freely. The cells treated with homocysteine were added to the collagen suspension. Gel diameters were measured by micrometers. Our experience with these cells suggests that ECs and SMCs tend to aggregate in collagen gel (42). Therefore, asymmetric contraction was observed. However, to minimize contribution due to asymmetric contraction, we measured diameter in two perpendicular directions and recorded the average of the two. Also, to enhance the reliability/reproducibility of the experiments, all the measurements of gel diameters were recorded in a blind fashion by a technician who was unaware of the experimental protocol. For the treatment with PPAR agonist, homocysteine and PPAR agonists were added to the cell suspension at the same time. Because homocysteine interacts with thiols in the proteins (17), it is possible that homocysteine modifies the collagen and induces conformational changes that affect the physical properties of collagen. We measured collagen gel diameters at 24 h in both the presence and absence of homocysteine (40 µM) and observed no difference in the diameters. All assays were repeated three times with triplicate wells per experimental condition.
Effects of EC, homocysteine, and PPAR agonist.
The collagen gels containing 105 ECs or SMCs with or
without homocysteine (40 µM) were prepared. The gel diameters were
measured at 24 h. To determine the optimal dose of homocysteine,
different concentrations of homocysteine were added. The effect of ECs
on SMC-mediated collagen gel contraction was measured by adding
104 ECs to 105 SMCs. The time dependence of
contraction was measured. The role of PPAR agonist was determined by
adding ciprofibrate (CF; Sigma Chemical) as PPAR- agonist and
15-deoxy-12,14-prostaglandin J2 (PGJ2;
CalBiochem) as PPAR-
agonist to the gel.
Analysis of DMP in SMCs.
To determine whether the contraction of SMCs induced by
homocysteine is mediated by a DMP, the expression of DMP in SMCs
treated with and without homocysteine was measured. Confluent cells
were deprived of serum for 24 h and were treated with DMEM
containing 0.2% FCS plus 40 µM homocysteine for 24 h. The
cell homogenate was analyzed for DMP by Western blot using DMP antibody
(Chemicon). The protein (25 µg/lane) was separated on a 10% SDS-PAGE
gel and was transferred onto a nitrocellulose membrane under reducing conditions. The nonspecific binding sites were blocked by 5% fat-free milk. The blots were developed with anti-DMP antibody (1:200) in PBS
containing 0.01% Tween. For -actin, Western blots were performed
using anti-mouse
-actin antibody (Sigma). The alkaline phosphatase-conjugated secondary antibody detection system was used.
The blots were scanned by Bio-Rad GS-700 densitometer.
Effects of DMP and cardiac inhibitor of metalloproteinase. To determine whether the blocking of DMP inhibits collagen gel contraction by SMCs, anti-DMP antibody (10 µg/ml) was added to the collagen gel suspension. To determine whether the inhibition of metalloproteinase activity of DMP modulates collagen gel contraction by SMCs, 20 µg/ml purified cardiac inhibitor of metalloproteinase (CIMP) (42) was added to the collagen gel suspension. The gel diameters were measured. CIMP is also known as TIMP-4. The expression of TIMP-4 in the heart is relatively higher than in any other tissue (11). We purified CIMP from rat hearts in our laboratory (42). Therefore, our findings suggest that CIMP plays a specific role in the heart.
Statistics. Assays were carried out in triplicate. Gel diameter and cell number are reported as means ± SD for each experiment. Experimental results were compared with controls without treatment under identical culture condition using Student's unpaired t-test. P < 0.05 was considered significant.
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RESULTS |
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Homocysteine-mediated collagen gel contraction.
Human aortic ECs and SMCs maintain their cobblestone and spindle-like
phenotype, respectively, for up to four and five passages. SMCs induce
contraction in collagen gel (i.e., decrease in gel diameter). Collagen
gel diameters at 0 and 24 h were 14.1 ± 0.2 and 14.0 ± 0.2 mm, respectively. Addition of homocysteine to SMCs further
increased collagen gel contraction (Fig.
1). The dose-response curve generated for
homocysteine-mediated collagen gel contraction suggests that a
pathophysiological concentration of homocysteine (20-60 µM)
induces significant contraction in collagen gel (Fig. 2A).
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Addition of ECs ameliorated SMC-mediated collagen gel contraction. The addition of ECs to SMCs decreased collagen gel contraction (i.e., increase in gel diameter) compared with SMCs alone. Homocysteine induced collagen gel contraction in SMCs in the presence of ECs (Fig. 2B). However, the contraction was smaller in SMCs plus ECs than in SMCs alone (Fig. 2B). These results suggest that factors released from ECs may inhibit the collagen gel contraction by SMCs.
Role of PPAR.
Addition of CF, an agonist of PPAR-, to the homocysteine plus SMC in
collagen gel did not affect the contraction. However, the addition of
PGJ2, an agonist of PPAR-
, decreased the
homocysteine-mediated SMC collagen gel contraction (Fig.
3A). The results suggest that PPAR-
regulates the SMC collagen gel contraction and that PPAR-
has no effect on contraction by SMCs (Fig. 3A). The
homocysteine also induced collagen contraction by ECs (Fig.
3B), but to a lesser degree than SMCs. The addition
of CF or PGJ2 to EC plus homocysteine in collagen gel
ameliorated the homocysteine-mediated collagen gel contraction. These
results suggest that PPAR-
and -
are involved in EC collagen
contraction. Both CF and PGJ2 inhibited the collagen gel
contraction induced by SMC plus ECs (Fig.
4), considering the fact that both
PPAR-
and -
are present on ECs and may modulate the SMC
contraction.
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Expression of DMP in SMCs.
To determine whether homocysteine induces DMP in SMCs, the levels of
DMP in cell homogenate were measured by Western blot analysis. The
results suggest that homocysteine increases DMP twofold in SMCs. The
cotreatment of homocysteine with 12 µM PGJ2 inhibits the
homocysteine-mediated DMP induction in SMCs (Fig. 5). These results suggest that
homocysteine induces DMP in SMCs by activation of PPAR-.
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Role of DMP and CIMP.
To determine whether DMP plays a role in SMC-mediated collagen gel
contraction, the SMCs were treated with anti-DMP antibody, or CIMP, in
collagen gel. Treatment with anti-DMP inhibited both contraction by
SMCs alone as well as contraction by homocysteine plus SMCs (Fig.
6A). Treatment with CIMP had
no effect on SMC contraction, although it inhibited the
homocysteine-mediated SMC collagen gel contraction (Fig.
6B). These results suggest that integrin may play an
important role in contraction by SMCs in the presence or absence of
homocysteine. However, metalloproteinase plays a significant role only
in homocysteine-mediated SMC collagen gel contraction.
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DISCUSSION |
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Toward the understanding of the mechanism by which
homocysteine mediates vascular constrictive remodeling, we have
demonstrated that homocysteine increases collagen gel contraction by
ECs as well as by SMCs. However, contraction by ECs is much less
significant than that by SMCs. The addition of ECs to SMCs ameliorates
the SMC-mediated contraction, but it is still higher than contraction by ECs alone. These results suggest that ECs partially inhibit SMC
collagen gel contraction and that they may be related to the endothelial nitric oxide (NO) generation. However, it is also possible that certain components of ECM remodeling, such as DMP, are
necessary for collagen contraction. Contraction is facilitated by
PPAR- and -
in ECs and by PPAR-
in SMCs. Homocysteine induces DMP in SMCs by activation of PPAR-
. Inhibition of integrin and metalloproteinase blocks homocysteine-mediated collagen gel contraction by SMCs.
Homocysteine impairs EC-dependent vasodilation (5) and induces vasoconstriction (32). To our knowledge, this is the first time that homocysteine has been shown to instigate vascular remodeling by contracting collagen gels with SMCs (Figs. 1 and 2). Previous results from our laboratory demonstrated an increase in SMC number after 24-h homocysteine treatment (45). It is likely that, during collagen contraction, SMC number is also increased. Although SMCs and ECs (Fig. 3) are known to induce collagen gel contraction separately (48), it was unclear whether ECs played any role in SMC-mediated collagen gel contraction. Our results demonstrate that ECs inhibit SMC-mediated collagen gel contraction (Fig. 2B). In pathogenesis of chronic homocysteinemia, a concentration of homocysteine in the 9-13 µM range induces hypertension and leads to chronic arteriosclerosis (39). However, levels of homocysteine of >20 µM decrease the incidence of survival by 35% due to coronary artery disease (31). Our results under in vitro, load-free conditions with higher doses of homocysteine may demonstrate a similar accelerated process of arteriosclerosis and a decrease in survival. However, the administration of fibrates ameliorates oxidative stress-mediated vascular dysfunction (9) and may improve the likelihood of survival.
The expression of a NAD(P)H oxidase subunit is studied in context of
the function of PPAR- and -
in ECs (15, 16, 33). However, the present study proposes an association between ECs and SMCs
in the induction of NAD(P)H oxidases and the development of oxidative
stress by homocysteine, which can be ameliorated by a putative effect
of PPAR-
and -
. The induction of PPAR in macrophages inhibits
matrix metalloproteinase-9 activity (24). It is unclear,
however, whether PPAR regulates homocysteine-mediated SMC vascular
remodeling. Our results suggest that PPAR-
and -
regulate the
endothelial response in SMC-mediated collagen gel contraction (Figs. 3
and 4). Although agonists of PPAR-
and PPAR-
induce
apoptosis in transformed ECs (13) or ECs
undergoing either tumorigenesis/angiogenesis (2) or
hepatocarcinogenesis (10), it is unclear whether
these agonists regulate normal EC and SMC phenotype. Collagen breaks
are required for opening of new integrin binding sites for cell
survival. However, a complete disconnect of cell and the ECM
leads to apoptosis. It is quite possible that these agonists
induce apoptosis in a cell of ECM disconnect. However, in
normal cells, these agonists increase cell survival.
Integrins are the primary cellular receptors for collagens, and an
antagonist of 1-integrin completely abolished contraction of
collagen gel by SMCs (21). Our results suggest that
homocysteine induces a DMP in SMCs (Fig. 5). Also, we have demonstrated
that DMP contributes significantly to collagen gel contraction. The inhibition of DMP by anti-DMP antibody blocks the SMC-mediated collagen
gel contraction (Fig. 6A). Remodeling implies synthesis and
degradation of ECM and SMC hypertrophy (43).
Metalloproteinases are increased in the vessel wall after injury
(51). Homocysteine causes redox injury in the vessel wall
and induces metalloproteinase activity (47). A
broad-spectrum inhibitor of metalloproteinase, batimastat, has been
shown to reduce the extent of wall shrinkage after angioplasty in pigs
(7). A metalloproteinase inhibitor has been shown to
decrease collagen gel contraction by fibroblasts (37). Our
results suggest that the inhibitor of metalloproteinase, CIMP, does not
block SMC contraction. However, CIMP blocks homocysteine-mediated collagen gel contraction (Fig. 6B), suggesting that
homocysteine-mediated collagen gel constriction is modulated by
metalloproteinase activity. Previously, we investigated the effect of
high homocysteine concentration on biomechanical characteristics of the
vasculature (i.e., passive distensibility and media-to-lumen ratio)
(30). The effects of homocysteine described in vitro in
the present study may result in vascular remodeling and narrowing of
the arterial/arteriolar lumen in vivo.
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ACKNOWLEDGEMENTS |
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This work was supported in part by National Institutes of Health Grants GM-48595 and HL-51971, American Heart Association-Mississippi Affiliate, and Kidney Care Foundation of Mississippi.
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FOOTNOTES |
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Address for reprint requests and other correspondence: S. C. Tyagi, Dept. of Physiology and Biophysics, Univ. of Mississippi Medical Center, 2500 North State St., Jackson, MS 39216-4505 (E-mail: styagi{at}physiology.umsmed.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published November 14, 2001;10.1152/ajpcell.00353.2001
Received 26 July 2001; accepted in final form 8 November 2001.
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