© 2000 American Heart Association, Inc.
Vascular Biology |
Rho-Kinase Is Involved in Macrophage-Mediated Formation of Coronary Vascular Lesions in Pigs In Vivo
Presented in part at the 72nd Scientific Sessions of the American Heart Association, Atlanta, Ga, November 7–10, 1999.
From the Department of Cardiovascular Medicine, Kyushu University Graduate School of Medical Sciences, Fukuoka, and the Division of Signal Transduction (K.K.), Nara Institute of Science and Technology, Ikoma, Japan.
Correspondence to Hiroaki Shimokawa, MD, PhD, Department of Cardiovascular Medicine, Kyushu University Graduate School of Medical Sciences, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. E-mail shimo{at}cardiol.med.kyushu-u.ac.jp
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Abstract |
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Abstract—We have previously shown that long-term treatment with an inflammatory cytokine from the adventitia causes the development of coronary vascular lesions, with the accumulation of macrophages. Recent studies in vitro have suggested that small G-protein Rho and its effector, Rho-kinase/ROK/ROCK, may be the key molecules for various cellular functions, including cell adhesion and movement. In this study, we examined whether adventitia-derived macrophages cause the formation of coronary vascular lesions in vivo and, if so, whether Rho-kinase is involved in the process. Porcine coronary segments from the adventitia were chronically treated with monocyte chemoattractant protein-1 alone, oxidized low density lipoprotein alone, or both. Vascular lesion formation (neointimal formation and development of vascular remodeling) was mostly enhanced at the coronary segment cotreated with monocyte chemoattractant protein-1 and oxidized low density lipoprotein, where the phosphorylation of myosin binding subunit of myosin phosphatase was increased, indicating an increased activity of Rho-kinase in vivo. Histological examination demonstrated that macrophages were accumulated at the adventitia and thereafter migrated into the vascular wall. Long-term oral treatment with fasudil, which is metabolized to a specific Rho-kinase inhibitor (hydroxyfasudil) after oral absorption, markedly inhibited the myosin binding subunit phosphorylation, the macrophage accumulation and migration, and the coronary lesion formation in vivo. These results indicate that Rho-kinase is involved in macrophage-mediated formation of coronary vascular lesions in our porcine model in vivo.
Key Words: macrophages • Rho-kinase • adventitia • arteriosclerosis • oxidative stress
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Introduction |
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Studies in vitro have suggested that monocyte recruitment into the vessel wall promoted by chemokines, including CC-chemokines, such as monocyte chemoattractant protein (MCP)-1, is an early step in the process of arteriosclerosis.1 2 3 Indeed, in mice lacking the receptor for MCP-1 (CCR2), the formation of atherosclerotic lesions is significantly attenuated.4 We have previously demonstrated that long-term local treatment with interleukin (IL)-1ß from the adventitia causes the development of coronary vascular lesions and vasospastic responses in pigs in vivo, with macrophage accumulation noted in the adventitia.5 6 7 Indeed, the importance of adventitial accumulation of inflammatory cells has been suggested for the pathogenesis of arteriosclerosis8 9 and acute coronary syndrome10 11 in general and for that of coronary lesion formation after coronary intervention12 13 in particular. However, it remains unknown whether adventitia-derived macrophages cause the formation of coronary lesions in vivo and, if so, what molecular mechanism(s) is involved in the process.
Recent studies in vitro have suggested that Rho family small G proteins, especially Rho and its effector, Rho-kinase/ROK/ROCK,14 15 16 17 may be the key molecules in various cellular functions, including vascular smooth muscle contraction and cell adhesion and movement.18 19 20 21 We also have recently demonstrated that the upregulation of Rho-kinase plays a key role in the pathogenesis of coronary artery spasm in our porcine model with IL-1ß,22 23 24 25 by inhibiting the myosin phosphatase and thereby enhancing the phosphorylations of myosin light chain in the coronary smooth muscle cells. Taken together, we hypothesized that the activation of Rho-kinase is involved not only in the vascular smooth muscle hypercontraction but also in the pathogenesis of arteriosclerosis.
Thus, the present study was designed to examine whether macrophages (accumulating in the adventitia) cause the coronary lesion formation in vivo and, if so, whether Rho-kinase activation is involved in the process. For this purpose, we used hydroxyfasudil, which we recently found is a specific inhibitor of Rho-kinase.23
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Methods |
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This experiment was reviewed by the Committee on Ethics in Animal Experiment of the Kyushu University and was carried out according to the Guidelines for Animal Experiments of the Kyushu University and of the Japanese Government.
Animal Preparation
Domestic male pigs (2 to 3 months old and weighing 25 to 30 kg,
Nihon Crea, Tokyo, Japan) were used. The animals were housed
individually under a controlled room temperature. They were sedated
with ketamine hydrochloride (12.5 mg/kg IM) and
anesthetized with sodium pentobarbital (20 mg/kg IV). They were
then intubated and mechanically ventilated with a respirator. Under
aseptic conditions, a left thoracotomy was performed, and the proximal
segments of the left anterior descending and left circumflex
coronary arteries were carefully
dissected.5 6 7
In the first series of experiments, 3 coronary segments were dissected and were gently wrapped with cotton mesh absorbing either recombinant human MCP-1 bound to microspheres (50 ng, Sigma Chemical Co) alone, oxidized LDL (Ox-LDL) alone (100 µg), or both (n=6). In other 6 animals, the dissected 3 coronary segments were treated with either MCP-1 alone, native LDL (100 µg) alone, or both. Animals were maintained on a regular diet for 4 weeks. We have previously confirmed that the treatment with control microspheres alone causes no significant lesions of the porcine coronary artery.5 Additional animals were cotreated with either a neutralizing mouse antibody to human MCP-1 (50 µg, Sigma; n=3) or nonimmune mouse IgG (50 µg, n=3), together with MCP-1 (50 ng), to confirm the in vivo effect of microsphere-bound MCP-1.
In the second series of experiments, as mentioned in detail in the following sections, histological examination was performed for the movement of adventitia-derived macrophages (n=6) and for immunostaining for macrophages and MCP-1 (n=6) 3, 7, and 14 days after the adventitial treatment with MCP-1 and Ox-LDL.
In the third series of experiments, an additional 12 animals received the adventitial treatment with MCP-1 and Ox-LDL at the 3 segments, and they were randomly divided into 2 groups: one group was treated with regular diet alone (control group, n=6), and another group was treated with oral administration of fasudil (5 mg/kg per day, Asahi Chemical Industry), starting 1 day before the operation and continuing for 2 weeks (fasudil group, n=6). The end point of 2 weeks was chosen because we noted in the first protocol series that the vascular lesion was already formed at 2 weeks after the surgery. The 3 coronary segments were randomly used for hematoxylin and eosin staining, immunostaining for macrophages (frozen section), and Western blot for phosphorylated myosin binding subunit (MBS). We recently found that fasudil is metabolized to hydroxyfasudil after oral absorption, which has a specific inhibitory effect on Rho-kinase; the Ki value (µmol/L) is 0.17 for Rho-kinase, 18 for protein kinase C, and 140 for myosin light chain kinase.23 We also confirmed that after oral absorption of fasudil, the effective serum concentration of hydroxyfasudil (>0.1 µmol/L) was maintained at least for 8 hours in pigs.
Preparation and Characterization of Ox-LDL
Native LDLs were isolated from human plasma by discontinuous
density gradient ultracentrifugation as described
previously.26 Briefly, the density of plasma was adjusted
to 1.006 g/mL with sodium chloride medium, and the plasma was
centrifuged at 150 000g for 24 hours. VLDL and the
chylomicron-rich layer were discarded. The remaining fraction, after
adjusting the density at 1.063 g/mL with potassium bromide medium, was
centrifuged at 150 000g for 24 hours to isolate LDL
from the HDL fraction. The purified LDL was dialyzed for 96 hours
against PBS, which was degassed with N2 and
contained 0.3 mmol/L EDTA, at 4°C. LDL was stored under
N2 at 4°C, and suitable aliquots were then
oxidized in the presence of 5 µmol/L CuSO4
for 18 to 20 hours at 37°C. Oxidation was terminated by
refrigeration. Oxidation of LDL was confirmed by the presence of
thiobarbituric acid–reactive substances, with malondialdehyde used as
a standard. Protein content was determined with BSA used as the
standard.26
Preparation of MCP-1 Microsphere Suspension
Microspheres (colored microspheres, E-Z Trac;
200 000 in number and 100 µm in diameter, which bind to the
amino residues of proteins, including cytokines) were added to
50 mL of 1 mmol/L HCl solution and centrifuged 4 times at
1200 rpm for 5 minutes. The microspheres were then resuspended
in 20 mL of NaHCO3/NaCl solution with 10 µg of
recombinant human MCP-1. The microspheres were allowed to bind
with MCP-1 at room temperature for 1 hour and then at 4°C overnight.
After centrifugation at 1200 rpm for 5 minutes, the
supernatant was separated. The MCP-1–bound microspheres in the
pellet were resuspended with Tris-HCl buffer solution for 1 hour to
block any remaining active sites.5 The MCP-1–bound
microspheres were finally washed and resuspended. The final
concentration of MCP-1 was 1 µg/mL, and 0.05 mL of the suspension (50
ng of MCP-1) was applied to the adventitia of the porcine
coronary artery. All of the above procedures were performed
under sterile conditions.5 With our method, MCP-1 is
firmly bound to microspheres by a covalent bond at the amino
residues of the protein and does not detach from the
spheres,5 allowing the detection of endogenous
MCP-1 by immunostaining in the coronary vessel
wall.
Fluorescent Microscopic Examination
Adventitia-derived macrophages were labeled in vivo
according to the method of Melnicoff et al.27 Briefly,
0.05 mL of fluorescent dye solution (1 µmol/L, PKH26
fluorescent staining kit, Zynaxis Cell Science) was
simultaneously applied at the adventitia with MCP-1 and
Ox-LDL, and the coronary artery was obtained 3, 7, and 14 days
after the procedure from different animals (n=2 each).
Fluorescent images were obtained by fluorescence
microscopy (LSM-GB200, Olympus) at an excitation wavelength of 551 nm
and an emission wavelength of 567 nm. In a preliminary study, we
confirmed that adventitia-derived macrophages absorb the
dye and become positive (by fluorescence examination) together
with elastic fibers (especially internal and external elastic lamina
[IEL and EEL, respectively]) and that circulating blood cells never
become positive by this method. Therefore, this method allows the
serial observation of the movement of adventitia-derived
macrophages in vivo.27
Histopathology and Immunohistochemistry
The animals were killed with a lethal dose of sodium
pentobarbital, and then the hearts were removed. Left coronary
arteries were perfused via a constant-pressure perfusion system (120 cm
H2O) with saline (500 mL) and subsequently with
5% formaldehyde (1000 mL).5 After the fixation, the left
anterior descending and left circumflex coronary arteries were
cut transversely, dehydrated, embedded in paraffin, and cut into
5-µm-thick slices. These sections were stained with hematoxylin and
eosin and van Gieson’s elastic stain for photomicroscopy. The degree
of intimal thickening was analyzed quantitatively with a
computer-assisted picture system (Genlocker System,
Sony).5
Three vessel areas were measured, including the luminal area and an area encircled by the IEL and EEL. The intimal area was determined as Ai=Ae-Al, where Ai is the intimal area, and Ae and Al are the areas within the IEL and the luminal area, respectively. The degree of neointimal formation was expressed by the following 3 parameters: intimal area (ie, Ai [mm2]), maximal intimal thickness (mm) measured with a caliper, and percent intima calculated by the following equation: Ai/Aex100 (%).5 6 7 The degree of vascular remodeling was expressed by the change in the 3 vessel areas by the following equation: (ATx-ACONT)/ACONTx100, where ATx and ACONT are vessel areas of the coronary segments at the treated and the normal control site, respectively.7 We used the average of 3 consecutive sections from a single arterial segment. The variability of the 3 sections was small.
In some experiments, these segments were embedded in OCT compound (Sakura Fine Technical Co) without being embedded in paraffin, cut into 5-µm-thick slices, and stained with oil red O. For immunostaining, these segments were immediately embedded in OCT compound without fixation, frozen, and cut into 5-µm-thick slices. Serial cryosections were stained with an antibody to human macrophages (PM-2K)28 or to rat MCP-1 (clone C4)29 or nonimmune mouse IgG (Zymed Laboratory). In a preliminary study, we confirmed that those antibodies cross-react well with porcine macrophages and MCP-1, respectively.
Measurements of Rho-Kinase Activity
To measure Rho-kinase activity, the extent of
phosphorylated MBS of myosin phosphatase, one of the
substrates of Rho-kinase,14 21 was measured by SDS-PAGE,
followed by electrophoretic transfer of the proteins to a
nitrocellulose membrane.25 The amounts of
phosphorylated MBS (20 µg protein in each sample)
were quantified by immunoblot procedures.25
ß-Actin was used as an internal control.
Data Analysis
All results are expressed as mean±SEM. Multiple comparisons
were made by ANOVA for repeated examinations, followed by the Fisher
post hoc test. Paired data were analyzed by Student
t test. A value of P<0.05 was considered to be
statistically significant.
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Results |
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Neointimal Formation and Geometric Remodeling of the Coronary Artery
At the coronary segment treated with MCP-1 alone or Ox-LDL alone, neointimal formation was noted, the extent of which was enhanced at the segment cotreated with MCP-1 and Ox-LDL (Figures 1A
and 1B). Similarly, the vascular
cross-sectional areas were reduced by those treatments compared with
the adjacent normal coronary segment (vascular remodeling), and
the extent of the remodeling was the highest at the segment cotreated
with MCP-1 and Ox-LDL (Figure 1C). No such augmenting effect was
noted with native LDL (n=6, data not shown). These vascular effects of
MCP-1 were abolished with the simultaneous treatment with a
neutralizing antibody against MCP-1 (n=3) but not with a nonimmune IgG
(n=3) (data not shown).
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Macrophage Migration Into the Coronary Artery
In Vivo
The histological examination with
fluorescent dye (Figure 2A) and
with an antibody to macrophages (Figure 2B) demonstrated
that a number of macrophages were accumulated at the adventitia
3 days after the operation and thereafter gradually migrated into the
vascular wall. There was no leakage of non–cell-associated dye in the
intima or the media 3 and 7 days after the procedure (Figure 2A). We confirmed that fluorescence-positive cells were
macrophages by immunostaining for the cells in
an adjacent section (Figure 2B). This migration of macrophages
was probably due in part to the expression of endogenous
MCP-1 in the media and the intima, as evidenced by the
immunostaining for the protein (Figure 2C). The
specificity of the immunostainings was confirmed by the
negative staining with nonimmune IgG (data not shown). Staining with
oil red O demonstrated that some of these adventitia-derived
macrophages caused foam cell lesions in the intima near the IEL
(data not shown).
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Rho-Kinase Activation
Immunoblot analysis demonstrated that the
Rho-kinase activity, as expressed by the
phosphorylation of MBS of myosin phosphatase after
normalization to ß-actin, was significantly increased at the segment
cotreated with MCP-1 and Ox-LDL by 
2.5 fold in the control group
(Figure 3).
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Inhibitory Effect of Fasudil/Hydroxyfasudil In
Vivo
Long-term oral treatment with fasudil inhibited the MBS
phosphorylation at the segment cotreated with MCP-1 and
Ox-LDL to the basal levels seen in the normal coronary artery
in the fasudil group, confirming its inhibitory effect on
Rho-kinase (Figure 3
). In the control group, the
macrophage accumulation was noted in the media and the
adventitia 2 weeks after the adventitial treatment with MCP-1 and
Ox-LDL; this accumulation was significantly inhibited in the fasudil
group (Figure 4). Finally, the long-term
treatment with fasudil significantly inhibited the
neointimal formation and the development of vascular
remodeling in vivo (Figure 5).
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Discussion |
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The novel findings of the present study were that (1) long-term adventitial treatment with MCP-1 induced neointimal formation and geometric remodeling of the coronary artery in vivo, (2) the formation of the coronary vascular lesions was enhanced by oxidative stress with Ox-LDL, (3) adventitia-derived macrophages played an important role in the creation of coronary vascular lesions in vivo, and (4) Rho-kinase was involved in the molecular mechanism for the formation of coronary vascular lesions in our porcine model in vivo. To the best of our knowledge, this is the first study that demonstrates the involvement of Rho-kinase in the macrophage-mediated formation of coronary vascular lesions in vivo.
Role of Adventitia-Derived Macrophages
Although previous studies have suggested the potential
importance of the adventitia,8 9 its role in vascular
lesion formation has been largely ignored. We have previously shown
that long-term adventitial treatment with inflammatory
cytokines causes the development of vascular remodeling,
neointimal formation, and vasospastic responses in porcine
coronary arteries in vivo.5 6 7 Because adventitial
accumulation of macrophages was noted in our models, we aimed
in the present study to examine the potential role of
adventitia-derived macrophages in vascular lesion formation in
vivo. To stimulate the adventitial accumulation of macrophages,
we chronically treated the adventitia of the porcine coronary
artery with MCP-1, a well-known CC-chemokine for
macrophages.1 2 Indeed, this treatment caused a
development of the vascular lesions similar to that found by us with
inflammatory cytokines.5 6 7 Furthermore, the
vascular lesion formation caused by MCP-1 was significantly enhanced by
cotreatment with Ox-LDL but not with native LDL, indicating an
enhancing effect of oxidative stress by Ox-LDL.30 31 It is
conceivable that macrophages were activated by Ox-LDL
and that they released cytokines and chemokines in the
adventitia, forming a vicious cycle toward a vascular lesion formation
in vivo. Although the number of macrophages in the intima was
relatively small even at 2 weeks after the application of MCP-1 and
Ox-LDL, we consider that the recruitment and activation of
macrophages at the adventitia per se initiated the
cytokine network in the media, resulting in the
neointimal formation and geometric remodeling in our
porcine model.
In the present study, we demonstrated the progressive migration of macrophages from the adventitia toward the intima. Although the exact mechanism of this macrophage migration remains to be clarified, the expression of endogenous chemokines (including MCP-1) may be involved because we showed the expression of endogenous MCP-1 in the intima and the media. It is also interesting to note that in the coronary segment cotreated with MCP-1 and Ox-LDL, foam cell lesions were developed in the intima in normolipemic animals. The interactions between the adventitia and the media/intima remain to be elucidated in future studies.
Involvement of Rho-Kinase Activation in Coronary
Lesion Formation
Rho-kinase has been implicated in the molecular mechanisms of not
only vascular smooth muscle contraction but also any phenomenon that
requires actomyosin interaction, including chemotaxis of inflammatory
cells.21 32 Furthermore, recent studies demonstrated that
Rho/Rho-kinase is substantially involved in the proliferation,
differentiation, adhesion, and migration of various types of cells,
including vascular smooth muscle cells and inflammatory
cells.19 33 Thus, it is highly possible that Rho-kinase is
involved in the molecular mechanism of
arteriosclerosis. Indeed, in the present study,
we demonstrated that Rho-kinase activity was significantly increased at
the coronary segment cotreated with MCP-1 and Ox-LDL.
Furthermore, hydroxyfasudil, which we recently found is a specific
inhibitor of Rho-kinase,23 markedly suppressed
the Rho-kinase activity (as evidenced by the Western blotting for
phosphorylated MBS) and inhibited the
macrophage accumulation in the adventitia, the migration into
the media, and the subsequent vascular lesion formation. We recently
observed that adenovirus-mediated transfer of dominant-negative
Rho-kinase significantly inhibits the neointimal formation
after balloon injury in porcine femoral arteries.34
Although macrophage accumulation at the adventitia was the
initial process of the coronary vascular lesion formation in
the present study, it is conceivable that Rho-kinase is
substantially involved in the pathogenesis of
arteriosclerosis, not only in macrophage
movement but also in many other processes.19 33 Thus,
Rho-kinase could be regarded as a novel target molecule for the
treatment of arteriosclerotic vascular
diseases.
Limitations of the Study
Several limitations of the present study can be raised. First,
it has been generally accepted that adhesion and migration of
monocytes/macrophages in the intima, rather than in the
adventitia, play an important role in the initiation of vascular lesion
formation.1 Although we also appreciate the importance of
the inflammatory changes in the intima, we consider that adventitial
inflammation may also play an important role in the vascular lesion
formation as discussed above.5 6 7 8 9 10 11 12 13 Furthermore, because we
found that focal and long-term treatment with MCP-1 is applicable at
the adventitia but not at the intima, we used the present
adventitial approach. Second, the doses of MCP-1 and of Ox-LDL that
were applied to the adventitia should have a clinical implication.
Indeed, the dose of MCP-1 corresponds to that found in the artery after
balloon injury.35 The dose of Ox-LDL was chosen on the
basis of a previous study that demonstrated the content of Ox-LDL in
atherosclerotic blood vessels.36 Third, the localization
of Rho-kinase in the coronary artery was not examined in the
present study. Although this point remains to be examined in a
future study, we consider from the data of macrophage migration
in vivo that one of the main sites for the Rho-kinase expression was
macrophages and, to a lesser extent, vascular smooth muscle
cells that showed hyperconstrictive response to
serotonin.23 24 25 Fourth,
endothelial function was not examined in the
present study. However, we have recently demonstrated that
endothelial vasodilator function is fairly preserved in
a porcine model with adventitial application of IL-1ß, an original
for the present model.37 Fifth, a detailed mechanism
for macrophage movement from the adventitia toward the intima
remains to be examined in future studies. Sixth, although we previously
confirmed that hydroxyfasudil has a specific inhibitory
effect on Rho-kinase,23 other effects of this compound
might be involved. This point remains to be examined in a future study
in which selective inhibition of Rho-kinase can be achieved by in vivo
gene transfer of dominant-negative Rho-kinase.34
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Acknowledgments |
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This work was supported in part by grants-in-aid from the Japanese Ministry of Health, Science, Sports and Culture, Tokyo, Japan. The authors wish to thank Dr M. Takeya, First Department of Pathology, Kumamoto University, for the generous gift of an antibody against macrophages and S. Tomita and E. Gunshima for their excellent technical assistance.
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Footnotes |
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Guest Editor for this article was Alan Fogelman, MD, UCLA Medical School, Los Angeles, Calif.
Received April 19, 2000; accepted July 20, 2000.
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