Kohtaro Abe

The Right Ventricle Under Pressure: Cellular and Molecular Mechanisms of Right-Heart Failure in Pulmonary Hypertension

Pulmonary arterial hypertension (PAH) is a deadly disease in which vasoconstriction and vascular remodeling both lead to a progressive increase in pulmonary vascular resistance. The response of the right ventricle (RV) to the increased afterload is an important determinant of patient outcome. Little is known about the cellular and molecular mechanisms that underlie the transition from compensated hypertrophy to dilatation and failure that occurs during the course of the disease. Moreover, little is known about the direct effects of current PAH treatments on the heart. Although the increase in afterload is the first trigger for RV adaptation in PAH, neurohormonal signaling, oxidative stress, inflammation, ischemia, and cell death may contribute to the development of RV dilatation and failure. Here we review cellular signaling cascades and gene expression patterns in the heart that follow pressure overload. Most data are derived from research on the left ventricle, but where possible specific information on the RV response to pressure overload is provided. This overview identifies the gaps in our understanding of RV failure and attempts to fill them, when possible. Together with the online supplement, it provides a starting point for new research and aims to encourage the pulmonary hypertension research community to direct some of their attention to the RV, in parallel to their focus on the pulmonary vasculature.

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.

Formation of Plexiform Lesions in Experimental Severe Pulmonary Arterial Hypertension

Background— The plexiform lesion is the hallmark of severe pulmonary arterial hypertension. However, its genesis and hemodynamic effects are largely unknown because of the limited availability of lung tissue samples from patients with pulmonary arterial hypertension and the lack of appropriate animal models. This study investigated whether rats with severe progressive pulmonary hypertension developed plexiform lesions. Methods and Results— After a single subcutaneous injection of the vascular endothelial growth factor receptor blocker Sugen 5416, rats were exposed to hypoxia for 3 weeks. They were then returned to normoxia for an additional 10 to 11 weeks. Hemodynamic and histological examinations were performed at 13 to 14 weeks after the Sugen 5416 injection. All rats developed pulmonary hypertension (right ventricular systolic pressure ≈100 mm Hg) and severe pulmonary arteriopathy, including concentric neointimal and complex plexiform-like lesions. There were 2 patterns of complex lesion formation: a lesion forming within the vessel lumen (stalk-like) and another that projected outside the vessel (aneurysm-like). Immunohistochemical analyses showed that these structures had cellular and molecular features closely resembling human plexiform lesions. Conclusions— Severe, sustained pulmonary hypertension in a very late stage of the Sugen 5416/hypoxia/normoxia-exposed rat is accompanied by the formation of lesions that are indistinguishable from the pulmonary arteriopathy of human pulmonary arterial hypertension. This unique model provides a new and rigorous approach for investigating the genesis, hemodynamic effects, and reversibility of plexiform and other occlusive lesions in pulmonary arterial hypertension.

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.

Acute vasodilator effect of fasudil, a Rho-kinase inhibitor, in monocrotaline-induced pulmonary hypertension in rats

Pulmonary arterial hypertension is a progressive and fatal disease for which Rho-kinase may be substantially involved. In this study, we examined the acute vasodilator effects of fasudil, a Rho-kinase inhibitor, in monocrotaline (MCT)-induced pulmonary hypertension (PH) in rats. Three weeks after a single subcutaneous injection of MCT (60 mg/kg), hemodynamic variables were measured under conscious and free-moving conditions before and after oral administration of fasudil. MCT caused a significant elevation of mean pulmonary arterial pressure (mPAP). Although a low dose of fasudil (3 mg/kg) had no effect on mPAP, a middle dose (10 mg/kg) caused a significant reduction in mPAP without change in mean systemic arterial pressure (mSAP), and a high dose (30 mg/kg) significantly reduced both mPAP and mSAP. Rho-kinase activity was significantly increased by MCT injection in pulmonary arteries but not in the aorta. Fasudil (10 mg/kg) inhibited only the Rho-kinase activity in pulmonary arteries without any effect in the aorta. Plasma concentration of hydroxyfasudil, a metabolite of fasudil, was within its clinical range in humans. These results demonstrate that fasudil exerts effective and selective vasodilatation of pulmonary arteries in rats with MCT-induced PH at a given dose, suggesting its usefulness for the treatment of the fatal 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.