Yasuhiro Eto

Rho-Kinase Is Involved in Macrophage-Mediated Formation of Coronary Vascular Lesions in Pigs In Vivo

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.

Long-term inhibition of Rho-kinase induces a regression of arteriosclerotic coronary lesions in a porcine model in vivo

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)-1beta. 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-1beta. Two weeks after the procedure, coronary stenotic lesions with constrictive remodeling and vasospastic response to serotonin were noted at the IL-1beta-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.

Long-Term Inhibition of Rho-Kinase Suppresses Neointimal Formation After Stent Implantation in Porcine Coronary Arteries: Involvement of Multiple Mechanisms

Objective—We recently demonstrated that Rho-kinase, an effector of the small GTPase Rho, is substantially involved in the pathogenesis of arteriosclerosis. In this study, we examined whether Rho-kinase is also involved in in-stent restenosis and if so, what mechanism is involved. Methods and Results—Pigs underwent stent implantation in the left coronary artery with or without administration of fasudil (30 mg/kg per day orally), a specific Rho-kinase inhibitor, starting 2 days before the procedure for a duration of 4 weeks. On day 28, reductions in coronary diameter and neointimal formation associated with macrophage accumulation, collagen deposition, and transforming growth factor (TGF)-&bgr;1 expression were noted at the stent site, and all were significantly suppressed by fasudil. On day 7, fasudil significantly increased the frequency of TUNEL-positive apoptotic cells, while it tended to reduce that of bromodeoxyuridine-positive proliferating cells in the neointima. Western blot analysis on day 7 demonstrated that phosphorylations of the ezrin/radixin/moesin family (a marker of Rho-kinase activity in vivo) and protein expression of monocyte chemoattractant protein-1and bcl-2 were upregulated at the stent site and were significantly suppressed by fasudil. Conclusions—These results indicate that long-term inhibition of Rho-kinase suppresses in-stent neointimal formation by multiple mechanisms, including reduced vascular inflammation, enhanced apoptosis, and decreased collagen deposition.

Adenovirus-Mediated Transfer of Dominant-Negative Rho-Kinase Induces a Regression of Coronary Arteriosclerosis in Pigs In Vivo

Abstract— Small GTPase Rho and its target Rho-kinase/ROK/ROCK play an important role in various cellular functions, including smooth muscle contraction, actin cytoskeleton organization, and cell adhesion and migration, all of which may be involved in the pathogenesis of arteriosclerosis. Here, we show that adenovirus-mediated transfer of dominant-negative Rho-kinase (DNRhoK) induces a marked regression of coronary constrictive remodeling and abolishes coronary vasospastic activity in vivo. Porcine coronary segments were chronically treated with interleukin-1&bgr;, which resulted in the development of constrictive remodeling and vasospastic responses to serotonin, as previously reported. Adenovirus-mediated transfer of DNRhoK, but not that of &bgr;-galactosidase, into the interleukin-1&bgr;-treated coronary segment caused a marked regression of the constrictive remodeling and abolished the vasospastic activity in 3 weeks. Western blot analysis showed that the phosphorylation of adducin and the ezrin/radixin/moesin family, the target proteins of Rho-kinase, were upregulated at the coronary lesions and were significantly suppressed by the transfer of DNRhoK. These results indicate that Rho-kinase is substantially involved in coronary constrictive remodeling and vasospastic responses, both of which can be reversed by the selective inhibition of the molecule in our porcine model in vivo.

Evidence for Protein Kinase C-Mediated Activation of Rho- Kinase in a Porcine Model of Coronary Artery Spasm

Objective—We have recently demonstrated that protein kinase C (PKC) and Rho-kinase play important roles in coronary vasospasm in a porcine model. However, it remains to be examined whether there is an interaction between the two molecules to cause the spasm. Methods and Results—A segment of left porcine coronary artery was chronically treated with IL-1&bgr;–bound microbeads in vivo. Two weeks after the operation, phorbol ester caused coronary spasm in vivo and coronary hypercontractions in vitro at the IL-1&bgr;–treated segment; both were significantly inhibited by hydroxyfasudil, a specific Rho-kinase inhibitor. Guanosine 5′-[&ggr;-thio]triphosphate (GTP&ggr;S), which activates Rho with a resultant activation of Rho-kinase, enhanced Ca2+ sensitization of permeabilized vascular smooth muscle cells, which were resistant to the blockade of PKC by calphostin C. The GTP&ggr;S-induced Ca2+ sensitization was greater in the spastic segment than in the control segment. Western blot analysis revealed that only PKC&dgr; isoform was activated during the hypercontraction. Conclusions—These results demonstrate that PKC and Rho-kinase coexist on the same intracellular signaling pathway, with PKC located upstream on Rho-kinase, and that among the PKC isoforms, only PKC&dgr; may be involved. Thus, the strategy to inhibit Rho-kinase rather than PKC may be a more specific and useful treatment for coronary spasm.

Overexpression of matrix metalloproteinase-9 promotes intravascular thrombus formation in porcine coronary arteries in vivo.

OBJECTIVE Matrix metalloproteinases (MMPs) cause extracellular matrix degradation and may be involved in the rupture of atherosclerotic plaques by degrading fibrous cap, resulting in the intravascular thrombus formation. Here we examined whether local overexpression of MMP-9 alters the characteristics of arteriosclerotic vascular lesions and promotes thrombosis after balloon injury in porcine coronary arteries in vivo. METHODS AND RESULTS Balloon angioplasty was performed in the left coronary arteries followed by injection of adenovirus vector solution encoding either MMP-9 or beta-galactosidase (beta-gal) gene into the injured coronary arteries. Three weeks after the gene transfer, histological examination demonstrated that macroscopic intravascular thrombus formation was noted at the MMP-9-transfected site but not at the beta-gal-transfected site. Microscopic intramural thrombus area was significantly larger at the MMP-9-transfected site as compared to the beta-gal-transfected site. Co-transfection of tissue inhibitor of metalloproteinase-1 (TIMP-1) with MMP-9 prevented the intravascular thrombus formation in vivo. Western blot analysis revealed the reduced expression of intact tissue factor pathway inhibitor-1 and the increased tissue factor (TF) expression at the MMP-9-transfected sites. CONCLUSION These results provide the first in vivo evidence that overexpression of MMP-9 promotes intravascular thrombus formation after balloon injury due in part to the activation of TF-mediated coagulation cascade.

Reduction in neointimal formation with a stent coated with multiple layers of releasable heparin in porcine coronary arteries.

Recent studies demonstrated that neointimal formation, which is caused by both neointimal proliferation and organized mural thrombus, is responsible for in-stent restenosis. Although various types of heparin coatings were effective in reducing (sub)acute thrombosis, most of them failed to reduce neointimal proliferation. This study was designed to examine the effect of the stent coated with multiple layers of releasable heparin complex from which heparin diffuses into the surrounding tissue and exerts its beneficial effects. Male Yorkshire pigs underwent balloon expandable stenting for coronary segments of both the left anterior and the left circumflex coronary arteries with a comparable diameter (n = 10). The stent implantation site was randomized for either control or heparin-coated stent. Four weeks after the procedure, quantitative coronary angiography (QCA) and intravascular ultrasonographic imaging (IVUS) were performed followed by histologic analysis. In additional animals, staining for proliferating cell nuclear antigen (PCNA) was performed 10 d after the procedure (n = 3). QCA demonstrated that coronary diameter (mm) was significantly larger at the heparin-coated stent site (2.32 ± 0.14) compared with the control stent site (1.81 ± 0.17) (p < 0.01). IVUS also showed that the neointimal area (mm2) was significantly suppressed at the heparin-coated stent site (2.12 ± 0.58) compared with the control stent site (3.92 ± 0.33) (p < 0.01). Histologic analysis also demonstrated that neointimal area (mm2) was significantly less at the heparin-coated stent (2.94 ± 0.43) than at the control stent site (4.41 ± 0.38) (p < 0.01), which was also the case for organized thrombus area (×10−4 mm2) (6.61 ± 2.67 vs. 19.36 ± 4.38, p < 0.01). The frequency of PCNA-positive vascular smooth muscle cells (%) was significantly less at the heparin-coated stent (10.8 ± 1.0) than at the control stent site (19.1 ± 1.7) (p < 0.01). These results suggest that the stent coated with releasable heparin is beneficial in reducing neointimal formation and subsequent in-stent restenosis.

Disparity of MCP-1 mRNA and protein expressions between the carotid artery and the aorta in WHHL rabbits: one aspect involved in the regional difference in atherosclerosis.

Objective—This study was designed to examine why in WHHL rabbits, muscular arteries, such as the carotid artery, are relatively resistant to atherosclerosis compared with the aorta, with a special reference to monocyte chemoattractant protein (MCP)-1. Methods and Results—MCP-1 mRNA expression was quantitated by Northern blot analysis, and its protein expression was quantitated by immunostaining and ELISA at the age of 1, 3, 6, and 12 months (n=5 to 6 each). In the aorta, atherosclerotic lesions were progressively developed with aging, and MCP-1 was highly expressed in endothelial cells and infiltrating macrophages. By contrast, in the carotid artery, atherosclerotic lesions and MCP-1 immunoreactivity were not evident throughout the experimental period. Unexpectedly, however, the extent of MCP-1 mRNA expression was comparable between the aorta and the carotid artery throughout the experimental period. Endothelial cells in primary culture from the aorta and the carotid artery expressed the same extent of MCP-1 mRNA on stimulation by oxidized LDL. There was no abnormality in primary structure of MCP-1 cDNA in WHHL. Conclusions—These results suggest that in WHHL, the atherosclerosis process, including MCP-1 protein expression, may be reduced in the carotid artery (and possibly in other muscular arteries), accounting in part for the regional resistance to atherosclerosis.

In vivo Gene Transfer of Dominant‐Negative Rho‐Kinase Induces Regression of Coronary Arteriosclerosis in Pigs

Abstract: Small GTPase Rho and its target Rho‐kinase play an important role in various cellular functions that may be involved in the pathogenesis of arteriosclerosis. Here we show that adenovirus‐mediated transfer of dominant‐negative Rho‐kinase (AdDNRhoK) induces a regression of coronary constrictive remodeling and abolishes coronary vasospastic activity in vivo. Porcine coronary segments were chronically treated with interleukin‐1β, which resulted in the development of constrictive remodeling and vasospastic responses to serotonin in vivo. AdDNRhoK, but not that of β‐galactosidase, into the interleukin‐1β‐treated coronary segment caused regression of constrictive remodeling and abolished vasospastic activity in 3 weeks. The unregulated phosphorylation of the target proteins of Rho‐kinase at the coronary lesion was significantly suppressed by AdDNRhoK. These results indicate that Rho‐kinase is substantially involved in the mechanism of coronary arteriosclerosis, which can be reversed by selective inhibition of the molecule in our porcine model in vivo.

Long-term treatment with propagermanium suppresses atherosclerosis in WHHL rabbits.

Macrophages play an important role in the pathogenesis of atherosclerosis, for which monocyte chemoattractant protein (MCP)-1 and CCR2 chemokine receptors may be involved. The authors have recently demonstrated that propagermanium exerts inhibitory effect on the CCR2 receptors. In the current study, the authors examined whether the organic germanium suppresses the MCP-1–induced monocyte migration in vitro and the development of atherosclerosis in WHHL rabbits in vivo. In the in vitro experiment, propagermanium concentration-dependently suppressed the MCP-1–induced migration of THP-1 cells. In the in vivo experiment, 20 WHHL rabbits were randomly divided into two groups; one group was treated with oral administration with propagermanium (9 mg/kg/day) for 3 months, and another group served as a control (n = 10 each). After 3 months, the aorta was isolated and stained with oil red O staining, and neointimal formation was quantified. Macrophage accumulation in the aorta was also evaluated by immunostaining. Long-term treatment with propagermanium did not affect the serum lipid profiles. However, the treatment significantly suppressed the oil red O–positive area of the total aorta (p < 0.05). Similarly, propagermanium significantly suppressed the intimal lesions (maximal intimal thickness and intimal area) and macrophage staining–positive area (all p < 0.05). A significant positive correlation was noted between macrophage staining–positive area and intimal lesions (p < 0.0001). These results indicate that long-term treatment with propagermanium suppresses the development of atherosclerosis in WHHL rabbits, suggesting its usefulness for the treatment of atherosclerotic vascular disease in humans.