Keiji Oi

Long-Term Treatment With a Rho-Kinase Inhibitor Improves Monocrotaline-Induced Fatal Pulmonary Hypertension in Rats

Abstract— Primary pulmonary hypertension is a fatal disease characterized by endothelial dysfunction, hypercontraction and proliferation of vascular smooth muscle cells (VSMCs), and migration of inflammatory cells, for which no satisfactory treatment has yet been developed. We have recently demonstrated that intracellular signaling pathway mediated by Rho-kinase, an effector of the small GTPase Rho, is involved in the pathogenesis of arteriosclerosis. In the present study, we examined whether the Rho-kinase–mediated pathway is also involved in the pathogenesis of fatal pulmonary hypertension in rats. Animals received a subcutaneous injection of monocrotaline, which resulted in the development of severe pulmonary hypertension, right ventricular hypertrophy, and pulmonary vascular lesions in 3 weeks associated with subsequent high mortality rate. The long-term blockade of Rho-kinase with fasudil, which is metabolized to a specific Rho-kinase inhibitor hydroxyfasudil after oral administration, markedly improved survival when started concomitantly with monocrotaline and even when started after development of pulmonary hypertension. The fasudil treatment improved pulmonary hypertension, right ventricular hypertrophy, and pulmonary vascular lesions with suppression of VSMC proliferation and macrophage infiltration, enhanced VSMC apoptosis, and amelioration of endothelial dysfunction and VSMC hypercontraction. These results indicate that Rho-kinase–mediated pathway is substantially involved in the pathogenesis of pulmonary hypertension, suggesting that the molecule could be a novel therapeutic target for the fatal disorder.

Extracorporeal Cardiac Shock Wave Therapy Markedly Ameliorates Ischemia-Induced Myocardial Dysfunction in Pigs in Vivo

Background—Prognosis of ischemic cardiomyopathy still remains poor because of the lack of effective treatments. To develop a noninvasive therapy for the disorder, we examined the in vitro and vivo effects of extracorporeal shock wave (SW) that could enhance angiogenesis. Methods and Results—SW treatment applied to cultured human umbilical vein endothelial cells significantly upregulated mRNA expression of vascular endothelial growth factor and its receptor Flt-1 in vitro. A porcine model of chronic myocardial ischemia was made by placing an ameroid constrictor at the proximal segment of the left circumflex coronary artery, which gradually induced a total occlusion of the artery with sustained myocardial dysfunction but without myocardial infarction in 4 weeks. Thereafter, extracorporeal SW therapy to the ischemic myocardial region (200 shots/spot for 9 spots at 0.09 mJ/mm2) was performed (n=8), which induced a complete recovery of left ventricular ejection fraction (51±2% to 62±2%), wall thickening fraction (13±3% to 30±3%), and regional myocardial blood flow (1.0±0.2 to 1.4±0.3 mL · min−1 · g−1) of the ischemic region in 4 weeks (all P<0.01). By contrast, animals that did not receive the therapy (n=8) had sustained myocardial dysfunction (left ventricular ejection fraction, 48±3% to 48±1%; wall thickening fraction, 13±2% to 9±2%) and regional myocardial blood flow (1.0±0.3 to 0.6±0.1 mL · min−1 · g−1). Neither arrhythmias nor other complications were observed during or after the treatment. SW treatment of the ischemic myocardium significantly upregulated vascular endothelial growth factor expression in vivo. Conclusions—These results suggest that extracorporeal cardiac SW therapy is an effective and noninvasive therapeutic strategy for ischemic heart 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.

Long-term inhibition of Rho-kinase ameliorates hypoxia-induced pulmonary hypertension in mice.

Pulmonary hypertension (PH) is a fatal disease characterized by endothelial dysfunction, hypercontraction and proliferation of vascular smooth muscle cells, and migration of inflammatory cells for which no satisfactory treatment has yet been developed. It has been recently demonstrated that Rho-kinase, an effector of the small GTPase Rho, is involved in the pathogenesis of arteriosclerosis and that long-term inhibition of Rho-kinase markedly ameliorates monocrotaline-induced PH in rats. However, it remains to be examined whether direct inhibition of Rho-kinase also ameliorates PH with a different etiology and whether endothelial nitric oxide synthase (eNOS) is involved in the beneficial effects of Rho-kinase inhibition. This study was designed to address those 2 important issues in a hypoxia-induced PH model using wild-type (WT) and eNOS-deficient (eNOS−/−) mice. Long-term blockade of Rho-kinase with fasudil (100 mg/kg/d) for 3 weeks markedly improved PH and right ventricular hypertrophy in WT mice with a lesser but significant inhibition noted in eNOS−/− mice. Fasudil upregulated eNOS with increased Akt phosphorylation in WT but not in eNOS−/− mice. These results suggest that long-term inhibition of Rho-kinase also ameliorates hypoxia-induced PH in mice, for which eNOS activation may partially be involved.

Application of Nanoparticle Technology for the Prevention of Restenosis After Balloon Injury in Rats

&NA; Restenosis after percutaneous coronary intervention continues to be a serious problem in clinical cardiology. Recent advances in nanoparticle technology have enabled us to deliver an antiproliferative drug selectively to the balloon‐injured artery for a longer time. NK911, which is a core‐shell nanoparticle of polyethyleneglycol‐based block copolymer encapsulating doxorubicin, accumulates in vascular lesions with increased permeability. We first confirmed that balloon injury caused a marked and sustained increase in vascular permeability (as evaluated by Evans blue staining) for a week in the rat carotid artery. We then observed that intravenous administration of just 3 times of NK911, but not doxorubicin alone, significantly inhibited the neointimal formation of the rat carotid artery at 4 weeks after the injury in both a single‐ and double‐injury model. Immunostaining demonstrated that the effect of NK911 was due to inhibition of vascular smooth muscle proliferation but not to enhancement of apoptosis or inhibition of inflammatory cell recruitment. Measurement of vascular concentrations of doxorubicin confirmed the effective delivery of the agent to the balloon‐injured artery by NK911 in both a single‐ and double‐injury model. RNA protection assay demonstrated that NK911 inhibited expression of several cytokines but not that of apoptosis‐related molecules. NK911 was well tolerated without any adverse systemic effects. These results suggest that nanoparticle technology to target vascular lesions with increased permeability is a promising and safe approach for the prevention of restenosis after balloon injury. The full text of this article is available at http://www.circresaha.org. (Circ Res. 2003;92:e62‐e69.)

Extracorporeal cardiac shock wave therapy improves left ventricular remodeling after acute myocardial infarction in pigs

ObjectiveWe have recently demonstrated that low-energy extracorporeal shock wave therapy improves chronic myocardial ischemia in pigs and humans. In this study, we examined whether our shock wave therapy is also effective at improving left ventricular remodeling after acute myocardial infarction in pigs. MethodsAcute myocardial infarction was created by surgically excising the proximal segment of the left circumflex coronary artery (n=20). In the early treatment protocol, the shock wave therapy was started 3 days after acute myocardial infarction, whereas in the late treatment protocol, the therapy was started 4 weeks after acute myocardial infarction (n=5 each). The remaining animals were treated in the same manner, but without the shock wave treatment in each protocol (n=5 each). ResultsIn the early treatment protocol, left ventricular ejection fraction was higher (42±1 vs. 32±1%, P<0.001) and left ventricular end-diastolic volume was smaller (95±1 vs. 99±2 ml, P<0.05) in the shock wave group compared with the control group. Furthermore, wall thickening fraction (32±1 vs. 28±1%, P<0.01), regional myocardial blood flow (1.7±0.2 vs. 1.0±0.1 ml/min/g, P<0.01), and number of capillaries in the border zone (1348±15 vs. 938±34 mm2, P<0.0001) were all significantly improved in the shock wave group compared with the control group. By contrast, in the late treatment group, no such beneficial effects of the shock wave therapy were noted. ConclusionThese results suggest that our extracorporeal cardiac shock wave therapy is also an effective and noninvasive therapy for improving left ventricular remodeling after acute myocardial infarction when started in the early phase of the disorder.

Remnant Lipoproteins From Patients with Sudden Cardiac Death Enhance Coronary Vasospastic Activity Through Upregulation of Rho-kinase

Objective—Sudden cardiac death (SCD) still remains a serious problem. We have previously shown that remnant-like particles (RLP) are the major risk factor for SCD and that Rho-kinase plays a central role in the molecular mechanism of coronary vasospasm. In this study, we examined whether RLP from patients with SCD upregulate Rho-kinase associated with an enhanced coronary vasospastic activity. Methods and Results—We isolated RLP and non-RLP in very-low-density lipoprotein (VLDL) fraction from SCD patients without coronary stenosis. We performed in vivo study in which we treated the coronary artery with RLP or non-RLP fraction at the adventitia in pigs. After 1 week, intracoronary serotonin caused marked coronary hyperconstriction at the segment treated with RLP fraction but not with non-RLP fraction (P <0.001, n=6), and hydroxyfasudil, a selective Rho-kinase inhibitor, dose-dependently inhibited the spasm in vivo. In organ chamber experiments, serotonin caused hypercontraction of vascular smooth muscle cells (VSMC) from RLP-treated segment, which was significantly inhibited by hydroxyfasudil (P <0.001, n=6). In cultured human coronary VSMC, the treatment with RLP significantly enhanced the expression and activity of Rho-kinase (P <0.05, n=6). Conclusions—These results indicate that RLP from SCD patients upregulate Rho-kinase in coronary VSMC and markedly enhance coronary vasospastic activity.

Prostacyclin does not inhibit rho-kinase: An implication for the treatment of pulmonary hypertension

Primary pulmonary hypertension continues to be a fatal disease. We have recently demonstrated that long-term inhibition of Rho-kinase, an effector of the small GTPase Rho, is effective for the treatment of pulmonary hypertension (PH) in rats and humans. Prostacyclin has been clinically used for the treatment of PH with moderate success. However, it remains to be examined whether Rho-kinase inhibition is involved in its beneficial effects on PH. In an ELISA assay, neither prostacyclin nor its oral analogue, beraprost sodium, inhibited Rho-kinase even at higher concentrations (10−7 to 10−5 M, 100 to 10,000 times higher than their clinical concentrations), whereas specific Rho-kinase inhibitors, fasudil and hydroxyfasudil, markedly (∼95%) inhibited the Rho-kinase activity at 10−5 M (near their clinical concentrations). Beraprost sodium did not significantly suppress serotonin-induced vascular smooth muscle cell (VSMC) contractions or Rho-kinase activity of the rat aorta without endothelium, as evaluated by the extent of phosphorylation of the ERM family, a substrate of Rho-kinase, whereas hydroxyfasudil markedly suppressed the VSMC contractions and Rho-kinase activity. These results indicate that prostacyclin lacks direct inhibitory effect on Rho-kinase and suggest that combination therapy with prostacyclin and a Rho-kinase inhibitor could exert further beneficial effects on PH.

In vivo magnetic resonance imaging of atherosclerotic lesions with a newly developed evans blue-DTPA-gadolinium contrast medium in apolipoprotein-E- deficient mice

Background: Magnetic resonance imaging (MRI) contrast agents that specifically detect atherosclerotic plaque may be useful for the noninvasive detection of the plaque. We have recently developed a new contrast agent, Evans blue-DTPA-gadolinium (EB-DTPA-Gd), which selectively accumulates vascular lesions with endothelial removal. In this study, we examined whether EB-DTPA-Gd is also useful for in vivo imaging of atherosclerotic plaques. Methods: We used male apolipoprotein-E-deficient (ApoE–/–) mice of different ages (3, 6 and 12 months old) and age-matched male wild-type mice. After a single intravenous administration of EB-DTPA-Gd (160 µM/kg body weight), MRI T1 signal was obtained in vivo. Results: Increased signal intensity in the aortic wall was noted within 10–20 min after intravenous injection of EB-DTPA-Gd and was maintained for 30 min. The MRI enhancement in the aorta of ApoE–/– mice was increased in accordance with age, whereas no such enhancement was noted in wild-type mice. Histological examination demonstrated that there was a topological correlation between the site of MRI enhancement and that of atherosclerotic plaque. Conclusions: These results indicate that EB-DTPA-Gd is a useful MRI contrast medium for the in vivo detection of atherosclerotic plaques.

Postprandial increase in plasma concentrations of remnant-like particles: an independent risk factor for restenosis after percutaneous coronary intervention.

Postprandial increase in remnant lipoprotein concentrations has been suggested as an important atherogenic factor. However, the influence of these remnants on the development of restenosis after percutaneous coronary intervention (PCI) remains to be examined. The present study was designed to address this point. In 60 consecutive patients with successful PCI, the influences of possible risk factors on the development of restenosis, including remnant-like particles (RLP) cholesterol (RLP-C) and triglyceride (RLP-TG), were examined. While mean concentrations of RLP-C and RLP-TG were normal in fasting state, postprandial change in RLP-C concentrations was a significant and independent risk factor for restenosis after PCI. The calculated cut-off index (COI) for the change was +64%. When the patients were divided into 2 groups according to this COI, minimal lumen diameter (MLD) and reference coronary diameter were comparable before and immediately after PCI between the high- (COI < 64%) and the low- (COI < 64%) responders. However, follow-up coronary angiography 3 to 6 months after PCI demonstrated that MLD, late loss, and loss index were all worse in the high responders compared with the low responders. These results indicate that post-prandial increase in RLP-C concentrations is an independent risk factor for restenosis after successful PCI, even in patients with normal fasting RLP-C levels.