© 2001 by European Society of Cardiology
Copyright © 2000, European Society of Cardiology
Long-term inhibition of Rho-kinase induces a regression of arteriosclerotic coronary lesions in a porcine model in vivo
aDepartment of Cardiovascular Medicine, Kyushu University Graduate School of Medical Sciences, 3-1-1, Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
bLife Science Institute, Asahi Chemical Co., Shizuoka, Japan
cNara Institute of Science and Technology, Ikoma, Japan
* Corresponding author. Tel.: +81-92-642-5360; fax: +81-92-642-5374 shimo{at}cardiol.med.kyushu-u.ac.jp
Received 15 December 2000; accepted 7 March 2001
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Abstract |
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 Top Abstract 1 Introduction2 Methods3 Results4 DiscussionReferences |
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Objective: We recently demonstrated that Rho-kinase/ROK/ROCK is functionally upregulated at the arteriosclerotic coronary lesions and plays a key role for coronary vasospastic responses in our porcine model with interleukin (IL)-1β. In the present study, we tested our hypothesis that Rho-kinase is involved in the pathogenesis of coronary arteriosclerosis per se in our porcine model. Methods: Segments of the left porcine coronary artery were chronically treated from the adventitia with IL-1β. Two weeks after the procedure, coronary stenotic lesions with constrictive remodeling and vasospastic response to serotonin were noted at the IL-1β-treated site, as previously reported. Then, animals were randomly divided into two groups; one group was treated with fasudil for 8 weeks followed by 1 or 4 weeks of washout period and another group served as a control. After oral absorption, fasudil is metabolized to hydroxyfasudil that is a specific inhibitor of Rho-kinase. Results: In the fasudil group, coronary stenosis and vasospastic response were progressively reduced in vivo, while the coronary hyperreactivity was abolished both in vivo and in vitro. Furthermore, Western blot analysis showed that in the fasudil group, the Rho-kinase activity (as evaluated by the extent of phosphorylation of myosin binding subunit of myosin phosphatase, one of the major substrates of Rho-kinase) was significantly reduced, while histological examination demonstrated a marked regression of the coronary constrictive remodeling. Conclusions: These results indicate that Rho-kinase is substantially involved in constrictive remodeling and vasospastic activity of the arteriosclerotic coronary artery, both of which could be reversed by long-term inhibition of the molecule in vivo. Thus, Rho-kinase may be regarded as a novel therapeutic target for arteriosclerotic vascular disease.
KEYWORDS Atherosclerosis; Cytokines; G-proteins; Remodeling; Signal transduction
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1 Introduction |
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TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
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Atherosclerosis is a slowly progressive inflammatory disease in which several mechanisms could be involved, including endothelial activation, migration of inflammatory cells and inflammatory/proliferative responses of vascular smooth muscle [1].
Recent studies in vitro and in vivo suggested that small G protein Rho and its target protein Rho-kinase/ROK/ROCK [2–4] play an important role for various cellular functions, including smooth muscle contraction [5,6], actin cytoskeleton organization [7,8], cell adhesion and migration [9], cytokinesis [10], and gene expression [5]. All these mechanisms could be involved in the pathogenesis of arteriosclerosis. We have recently demonstrated that Rho-kinase is functionally upregulated in our porcine model with IL-1β, playing a key role for coronary artery spasm by enhancing calcium sensitivity of vascular smooth muscle through inhibition of myosin phosphatase (MLCPh) [11]. Coronary lesions in our porcine model are characterized by constrictive remodeling, mild intimal thickening and vasospastic responses [12,13], which are similar to those observed in humans. Thus, our porcine model may be useful to examine the molecular mechanism of coronary arteriosclerosis in humans.
We also recently demonstrated that hydroxyfasudil, an active metabolite of fasudil after oral absorption, is a potent and specific inhibitor of Rho-kinase and markedly inhibits coronary hyperconstriction in our porcine model [14]. Recent studies suggested that Rho-kinase may play an important role in the signal transduction initiated by angiotensin II [15], thrombin [16], endothelin-1 [17], and serotonin [11,14].
In the present study, we thus examined our hypothesis that Rho-kinase is substantially involved in the pathogenesis of arteriosclerotic coronary lesions with constrictive remodeling and vasospastic activity in our porcine model in vivo.
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2 Methods |
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This research was reviewed by the Committee of the Ethics on Animal Experiments at Kyushu University and was carried out in accordance with the Guidelines for Animal Experiment at Kyushu University and that of the Japanese Government.
2.1 Animal preparation
Domestic male pigs (2–3 months old, and weighing 25–30 kg) (Nihon Crea, Tokyo, Japan) were sedated with ketamine hydrochloride (1.5 mg/kg i.m.) and were then anesthetized with sodium pentobarbital (30 mg/kg i.v.). The animals were then intubated and ventilated with room air via positive-pressure ventilator (Shinano, Tokyo). Under aseptic conditions, a left thoracotomy was performed, and the proximal segments of the left anterior descending and the left circumflex coronary arteries with the same external diameter were carefully dissected. Three segments were usually dissected in the two left coronary arteries (Fig. 1). The dissected segments were applied from the adventitia with recombinant human IL-1β beads suspension (2.5 µg in 0.05 ml) absorbed in cotton mesh as previously described [12].
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Two weeks after the operation, animals were anesthetized in the same manner, and then the right carotid artery was surgically exposed, and a 10 F sheath was inserted. After systemic heparinization (10 000 U/body), a preshaped Judkin's catheter (10 F, Medtronic, Minneapolis) was inserted into the carotid artery and coronary angiography in a left anterior oblique view was performed using the cineangiography system (Toshiba Medical, Tokyo). Throughout the procedure, ECGs in leads I, II, III, V1, and V6 were recorded (NEC San-Ei Polygraph System, Tokyo) and the arterial pressure was measured with a pressure transducer (Gould, Cleveland) connected to a Judkin's catheter. After coronary angiography was performed, the carotid artery was ligated, the skin closed, and the animals allowed to recover from anesthesia.
Then, the animals were randomly divided into two groups; one group was treated with oral administration of fasudil (5 mg/kg/day) for 8 weeks followed by 1 or 4 weeks of washout period, while another group served as a control. We previously confirmed that fasudil is metabolized to hydroxyfasudil after oral absorption (<95%), which has a specific inhibitory effect of Rho-kinase; the ki value (µM) is 0.17 for Rho-kinase, 18 for protein kinase C, and 140 for myosin light chain kinase [14]. The washout period was set in order to avoid the direct effect of hydroxyfasudil remained either in the blood or in the blood vessels on coronary vasomotion. Coronary angiography was repeated in the same manner at 4, 8 (after 1-day washout period), and 9 weeks (after 1-week washout period) after the randomization (Fig. 2). In some animals, coronary angiography was again repeated 12 weeks (after 4-week washout period) after the randomization. After the in vivo experiments, animals were killed with an excess dose of pentobarbital (60–90 mg/kg i.v.) and coronary arteries were excised. The IL-1β-treated coronary segments were used, in a randomized manner, for histological examination (with perfusion fixation), organ chamber experiment, and Western blotting (Fig. 1).
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2.2 Preparation of IL-1β beads
IL-1β beads suspension was prepared as previously described in detail [12].
2.3 Coronary angiography and coronary diameter measurement
Coronary angiography was performed in a left anterior oblique view as previously described [11–14]. The extent of coronary stenosis at the IL-1β-treated segments was expressed as the percent decrease in the luminal diameter compared to the mean diameter of adjacent proximal and distal normal coronary segments after nitroglycerin (10 µg/kg i.c.). The extent of coronary constriction induced by intracoronary serotonin (10 µg/kg) or prostaglandin F2
(PGF2, 50 µg/kg) was expressed as a percent decrease in the luminal diameter from the control level [11–14].
2.4 Organ chamber experiment
Vascular reactivity was also examined in vitro in organ chamber experiments using isolated coronary arteries from the IL-1β-treated and the normal segments, as previously described [11,14,18]. The extent of serotonin (1 µmol/l)-induced contraction was expressed as a percentage of the contraction induced by 62 mmol/l KCl [11,14,18].
2.5 Measurement of Rho-kinase activity
To evaluate Rho-kinase activity, the extent of Rho-kinase-dependent phosphorylation of myosin binding subunit (MBS) was quantified by Western blot analysis, using specific antibody against MBS phosphorylated at Ser-854 [19], as previously described [11].
2.6 Histological examination
After the in vivo experiment, the heart was removed and one of the left coronary arteries was perfused with 6% formalin at a pressure of 120 mmHg and fixed with formalin for a week [12]. For the light microscopic examination, tissue samples were embedded in paraffin, sectioned into slices 5 µm thick, mounted on glass slides, and stained with hematoxylin–eosin and van Gieson's methods. With a photomicroscopic photograph system (Nikon, Tokyo), pictures were taken at x20 and x40 magnifications. In each specimen, the area encircled by the lumen, IEL and EEL was calculated with an automated computer-based image analyzer (KD4600, Graphic Corp, Yokohama, Japan). Coronary constrictive remodeling was assessed by measuring the ratio of the areas (EEL, IEL, and lumen areas) at the IL-1β-treated coronary segments to the mean value of those areas of adjacent proximal and distal normal coronary segments, respectively.
2.7 Data analysis
All results were measured by experienced observers blinded to the origin of the samples, and expressed as the mean±S.E.M. Multiple comparisons were made by ANOVA for repeated examinations followed by Fisher's post-hoc test. Paired data were analyzed by Student's t-test. A P value of <0.05 was considered to be statistically significant.
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3 Results |
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3.1 Coronary angiography
Blood pressure and heart rate did not significantly change in both groups throughout the experimental period (data not shown). Two weeks after the IL-1β application, coronary angiography confirmed the development of stenotic lesion and vasospastic response to serotonin at the IL-1β-treated coronary segments as previously described [11–14]. Serial coronary angiography revealed that the extent of coronary stenosis and vasospastic response to serotonin was progressively reduced in the fasudil-treated but not in the control group (Figs. 1 and 2
). By contrast, the inhibitory effect of the fasudil treatment on the serotonin-induced contraction was not evident at the control site (Figs. 1 and 2). The extent of coronary vasoconstriction to intracoronary PGF2 was comparable between the two groups throughout the experimental period as previously reported (data not shown) [12,14,18]. Importantly, no rebound phenomenon of the vasospastic response to serotonin was noted at 1 week (n=8, Fig. 2) and even 4 weeks after (n=3, data not shown) the cessation of the fasudil treatment.
3.2 Organ chamber experiment
In the control group, serotonin-induced contraction of the IL-1β-treated coronary segment was significantly greater compared with the normal segment, whereas in the fasudil group, such hypercontraction of the IL-1β-treated segment was no longer noted even after the washout of the inhibitor (Fig. 3).
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3.3 Measurement of Rho-kinase activity
Western blot analysis demonstrated that in the fasudil group, the Rho-kinase-dependent phosphorylation of MBS upon stimulation by serotonin (1 µmol/l) was significantly increased in the IL-1β-treated coronary segment compared with the normal coronary segment as previously reported (Fig. 4) [11,14]. Importantly, this increase in the MBS phosphorylation was significantly suppressed in the fasudil group even after the washout of the inhibitor (Fig. 4).
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3.4 Histological examination
Histological examination demonstrated that the EEL, IEL and lumen areas were all significantly reduced at the IL-1β-treated segments in the control group, and this constrictive remodeling markedly regressed in the fasudil group (Figs. 5 and 6
). Neointimal formation was minimal in both groups, indicating that the improvement of coronary luminal narrowing in the fasudil group was caused primarily by the regression of constrictive remodeling (Figs. 5 and 6).
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4 Discussion |
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The novel finding of the present study was that the long-term inhibition of Rho-kinase with hydroxyfasudil caused a marked regression of coronary constrictive remodeling and an abolishment of coronary vasospastic activity in our porcine model with IL-1β in vivo. These results provide the first evidence that Rho-kinase is substantially involved in constrictive remodeling and vasospastic activity of the arteriosclerotic coronary artery, both of which could be reversed by the long-term inhibition of the molecule in vivo.
4.1 Rho-kinase and coronary arteriosclerosis
We have recently demonstrated that the expression of Rho-kinase is upregulated in our porcine model with IL-1β, which accounts, at least in part, for the Rho-kinase-dependent inhibition of MLCPh and the resultant increase in calcium sensitivity of vascular smooth muscle [11–14,18,20]. Recent studies in vitro showed that Rho-kinase and MLCPh coordinate the phosphorylation state of ERM family (ezrin/radixin/moesin) and adducin, target molecules of Rho-kinase, all of which may play an important role in cell adhesion and migration [21,22]. Indeed, Rho-kinase and its target molecules mediate actin cytoskeleton organization [7,8], cell to cell adhesion [9], cell to extracellular matrix adhesion [9], cell migration [9], cytokinesis [10] and gene expression [5]. Some of these mechanisms are regulated with adhesion complexes that consist of focal adhesion, anchoring proteins and actin filament [9]. We have recently observed that the expression of the ERM family and adducin is also upregulated at the IL-1β-treated segment in our porcine model (unpublished observations). Furthermore, Rho-kinase may play an important role in the signal transduction initiated by angiotensin II [15], thrombin [16], endothelin-1 [17], and serotonin [11,14]. Thus, it is possible that the upregulated Rho-kinase causes coronary arteriosclerotic lesions in our porcine model through activation of those target molecules and vasoactive substances. Although the molecular mechanism for the upregulation of Rho-kinase in our porcine model remains to be elucidated, it was recently reported that IL-1β enhances Rho-kinase activity in vitro [23]. Furthermore, in our porcine model, application of IL-1β may activate a cytokine network that consists of various inflammatory cytokines and growth factors, including tumor necrosis factor- [13], platelet-derived growth factor (PDGF) [12], and fibroblast growth factor-1 [24]. We are currently working on the molecular mechanism for the transcriptional regulation of Rho-kinase.
4.2 Long-term inhibition of Rho-kinase causes a regression of coronary constrictive remodeling
One of the novel findings of this study was that the long-term inhibition of Rho-kinase caused a marked regression of constrictive remodeling of the coronary artery in vivo (Figs. 1, 2, 5 and 6). In our porcine model with IL-1β, the arteriosclerotic coronary lesions with vasospastic activity persist for at least 3 months after the application of the cyotokine [12]. Our finding suggests that coronary constrictive remodeling could be a reversible phenomenon in which Rho-kinase is substantially involved in our porcine model. It is conceivable that this regression of coronary remodeling was caused in part by the inhibition of the target molecules of Rho-kinase (e.g. ERM family and adducin) as they mediate various cellular functions that may be important for the maintenance of constrictive remodeling [5,8,21,22]. We recently demonstrated that Rho-kinase is functionally upregulated by balloon injury and that the subsequent vascular lesion formation is suppressed by the gene transfer of dominant-negative Rho-kinase in porcine femoral arteries in vivo [25]. We also recently shown that long-term treatment with hydroxyfasudil suppresses coronary vascular lesion formation caused by macrophages in porcine coronary arteries in vivo [26]. Similarly, it was recently reported that long-term treatment with Y-27632, another inhibitor of Rho-kinase, suppresses the vascular lesion formation after balloon injury in rat carotid arteries [27]. Among the cellular functions mediated by Rho-kinase and its target molecules, cell adhesion may be important for the regulation of three-dimensional structure of blood vessels including vascular remodeling [28].
Geometric (constrictive) remodeling is important for the luminal narrowing after coronary angioplasty [29,30]. We previously demonstrated that adventitial inflammatory/proliferative responses play an important role in the pathogenesis of geometric remodeling in porcine coronary arteries in vivo because adventitial delivery of antiproliferative agents (such as a tyrosine kinase inhibitor) was effective to prevent the vascular lesion formation [31,32]. Recent studies indicate that collagen accumulation in the vessel wall and endothelial dysfunction are both associated with constrictive remodeling [33]. Since endothelial vasodilator function is fairly preserved in our porcine model [34], it is possible that alterations of smooth muscle properties with collagen accumulation play a major role in the pathogenesis of constrictive remodeling in our porcine model.
4.3 Long-term inhibition of Rho-kinase abolishes coronary vasospastic activity
Another novel finding of this study was that the long-term blockade of Rho-kinase with hydroxyfasudil abolished the vasospastic activity of the arteriosclerotic coronary artery in association with the marked regression of coronary constrictive remodeling (Figs. 1–3). This was not due to acute effect of hydroxyfasudil or direct geometric effect of the coronary artery because no vasospastic activity was noted even after the washout of the inhibitor (Figs. 2 and 3) and because the coronary vasoconstricting response to PGF2 was unaltered with the regression of the remodeling, respectively. These results suggest that coronary vasospastic activity could also be reversed by the long-term inhibition of Rho-kinase. Although the molecular mechanism for this observation remains to be examined, it is possible that the long-term inhibition of Rho-kinase resulted in the downregulation of the molecule itself, its target molecules and other related molecules that are involved in both the vasospastic activity and the constrictive remodeling of the coronary artery. The present finding is clinically important because calcium channel blockers cannot abolish coronary vasospastic activity per se as evidenced by the rebound coronary spasm after abrupt cessation [35].
4.4 Limitations of the study
Several limitations can be raised for the present study. First, this study was performed in a porcine model of coronary arteriosclerosis but not in humans. Thus, the present observations need to be confirmed in a future study in patients with coronary artery disease. Second, although hydroxyfasudil is a specific inhibitor of Rho-kinase, it also has a weak inhibitory effect on other kinases (e.g. protein kinase C, myosin light chain kinase) [14]. However, we have recently demonstrated that in vivo gene transfer of dominant-negative Rho-kinase also induces a marked regression of coronary constrictive remodeling and an abolishment of coronary vasospastic activity in our porcine model [36]. Third, the possible toxic effects of the long-term administration of fasudil/hydroxyfasudil should be clarified. This point is important especially from a clinical point of view. Our preliminary study and the late phase II trial of fasudil for angina pectoris in Japan showed that no major complications were noted with the treatment (unpublished observations). Furthermore, the present study also showed that the treatment did not significantly affect the vasomotion of the normal coronary artery.
In summary, the present study demonstrated that the long-term inhibition of Rho-kinase causes a marked regression of coronary constrictive remodeling and an abolishment of coronary vasospastic activity in our porcine model, suggesting that Rho-kinase may be regarded as a novel therapeutic target for arteriosclerotic vascular disease.
Time for primary review 27 days.
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Acknowledgements |
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We thank T. Akiyama and I. Kunihiro for their cooperation, and S. Tomita, E. Gunshi-ma, and M. Sonoda for their excellent technical assistance. This work was supported in part by grants from the Ministry of Education, Science, Sports and Culture, Tokyo, Japan (07457113, 09281225, 09470169, 10177223, 10357006, 12032215, 12470158).
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