Ryo Kurosawa

Basigin Mediates Pulmonary Hypertension by Promoting Inflammation and Vascular Smooth Muscle Cell Proliferation

Rationale: Cyclophilin A (CyPA) is secreted from vascular smooth muscle cells (VSMCs) by oxidative stress and promotes VSMC proliferation. However, the role of extracellular CyPA and its receptor Basigin (Bsg, encoded by Bsg) in the pathogenesis of pulmonary hypertension (PH) remains to be elucidated. Objective: To determine the role of CyPA/Bsg signaling in the development of PH. Methods and Results: In the pulmonary arteries of patients with PH, immunostaining revealed strong expression of CyPA and Bsg. The pulmonary arteries of CyPA+/– and Bsg+/– mice exposed to normoxia did not differ in morphology compared with their littermate controls. In contrast, CyPA+/– and Bsg+/– mice exposed to hypoxia for 4 weeks revealed significantly reduced right ventricular systolic pressure, pulmonary artery remodeling, and right ventricular hypertrophy compared with their littermate controls. These features were unaltered by bone marrow reconstitution. To further evaluate the role of vascular Bsg, we harvested pulmonary VSMCs from Bsg+/+ and Bsg+/– mice. Proliferation was significantly reduced in Bsg+/– compared with Bsg+/+ VSMCs. Mechanistic studies demonstrated that Bsg+/– VSMCs revealed reduced extracellular signal–regulated kinase 1/2 activation and less secretion of cytokines/chemokines and growth factors (eg, platelet-derived growth factor-BB). Finally, in the clinical study, plasma CyPA levels in patients with PH were increased in accordance with the severity of pulmonary vascular resistance. Furthermore, event-free curve revealed that high plasma CyPA levels predicted poor outcome in patients with PH. Conclusions: These results indicate the crucial role of extracellular CyPA and vascular Bsg in the pathogenesis of PH.

Protective Roles of Endothelial AMP-Activated Protein Kinase Against Hypoxia-Induced Pulmonary Hypertension in Mice

RATIONALE Endothelial AMP-activated protein kinase (AMPK) plays an important role for vascular homeostasis, and its role is impaired by vascular inflammation. However, the role of endothelial AMPK in the pathogenesis of pulmonary arterial hypertension (PAH) remains to be elucidated. OBJECTIVE To determine the role of endothelial AMPK in the development of PAH. METHODS AND RESULTS Immunostaining showed that endothelial AMPK is downregulated in the pulmonary arteries of patients with PAH and hypoxia mouse model of pulmonary hypertension (PH). To elucidate the role of endothelial AMPK in PH, we used endothelial-specific AMPK-knockout mice (eAMPK(-/-)), which were exposed to hypoxia. Under normoxic condition, eAMPK(-/-) mice showed the normal morphology of pulmonary arteries compared with littermate controls (eAMPK(flox/flox)). In contrast, development of hypoxia-induced PH was accelerated in eAMPK(-/-) mice compared with controls. Furthermore, the exacerbation of PH in eAMPK(-/-) mice was accompanied by reduced endothelial function, upregulation of growth factors, and increased proliferation of pulmonary artery smooth muscle cells. Importantly, conditioned medium from endothelial cells promoted pulmonary artery smooth muscle cell proliferation, which was further enhanced by the treatment with AMPK inhibitor. Serum levels of inflammatory cytokines, including tumor necrosis factor-α and interferon-γ were significantly increased in patients with PAH compared with healthy controls. Consistently, endothelial AMPK and cell proliferation were significantly reduced by the treatment with serum from patients with PAH compared with controls. Importantly, long-term treatment with metformin, an AMPK activator, significantly attenuated hypoxia-induced PH in mice. CONCLUSIONS These results indicate that endothelial AMPK is a novel therapeutic target for the treatment of PAH.

Basigin Promotes Cardiac Fibrosis and Failure in Response to Chronic Pressure Overload in Mice

Objective— Basigin (Bsg) is a transmembrane glycoprotein that activates matrix metalloproteinases and promotes inflammation. However, the role of Bsg in the pathogenesis of cardiac hypertrophy and failure remains to be elucidated. We examined the role of Bsg in cardiac hypertrophy and failure in mice and humans. Approach and Results— We performed transverse aortic constriction in Bsg +/– and in wild-type mice. Bsg +/– mice showed significantly less heart and lung weight and cardiac interstitial fibrosis compared with littermate controls after transverse aortic constriction. Both matrix metalloproteinase activities and oxidative stress in loaded left ventricle were significantly less in Bsg +/– mice compared with controls. Echocardiography showed that Bsg +/– mice showed less hypertrophy, less left ventricular dilatation, and preserved left ventricular fractional shortening compared with littermate controls after transverse aortic constriction. Consistently, Bsg +/– mice showed a significantly improved long-term survival after transverse aortic constriction compared with Bsg +/+ mice, regardless of the source of bone marrow (Bsg +/+ or Bsg +/– ). Conversely, cardiac-specific Bsg-overexpressing mice showed significantly poor survival compared with littermate controls. Next, we isolated cardiac fibroblasts and examined their responses to angiotensin II or mechanical stretch. Both stimuli significantly increased Bsg expression, cytokines/chemokines secretion, and extracellular signal–regulated kinase/Akt/JNK activities in Bsg +/+ cardiac fibroblasts, all of which were significantly less in Bsg +/– cardiac fibroblasts. Consistently, extracellular and intracellular Bsg significantly promoted cardiac fibroblast proliferation. Finally, serum levels of Bsg were significantly elevated in patients with heart failure and predicted poor prognosis. Conclusions— These results indicate the crucial roles of intracellular and extracellular Bsg in the pathogenesis of cardiac hypertrophy, fibrosis, and failure in mice and humans.

Low-Intensity Pulsed Ultrasound Enhances Angiogenesis and Ameliorates Left Ventricular Dysfunction in a Mouse Model of Acute Myocardial Infarction

Objective— Left ventricular (LV) remodeling after acute myocardial infarction still remains an important issue in cardiovascular medicine. We have recently demonstrated that low-intensity pulsed ultrasound (LIPUS) therapy improves myocardial ischemia in a pig model of chronic myocardial ischemia through enhanced myocardial angiogenesis. In the present study, we aimed to demonstrate whether LIPUS also ameliorates LV remodeling after acute myocardial infarction and if so, to elucidate the underlying molecular mechanisms involved in the beneficial effects of LIPUS. Approach and Results— We examined the effects of LIPUS on LV remodeling in a mouse model of acute myocardial infarction, where the heart was treated with either LIPUS or no-LIPUS 3 times in the first week (days 1, 3, and 5). The LIPUS improved mortality and ameliorated post–myocardial infarction LV remodeling in mice. The LIPUS upregulated the expression of vascular endothelial growth factor, endothelial nitric oxide synthase, phosphorylated ERK, and phosphorylated Akt in the infarcted area early after acute myocardial infarction, leading to enhanced angiogenesis. Microarray analysis in cultured human endothelial cells showed that a total of 1050 genes, including those of the vascular endothelial growth factor signaling and focal adhesion pathways, were significantly altered by the LIPUS. Knockdown with small interfering RNA of either &bgr;1-integrin or caveolin-1, both of which are known to play key roles in mechanotransduction, suppressed the LIPUS-induced upregulation of vascular endothelial growth factor. Finally, in caveolin-1–deficient mice, the beneficial effects of LIPUS on mortality and post–myocardial infarction LV remodeling were absent. Conclusions— These results indicate that the LIPUS therapy ameliorates post–myocardial infarction LV remodeling in mice in vivo, for which mechanotransduction and its downstream pathways may be involved.

PDE1C Negatively Regulates Growth Factor Receptor Degradation and Promotes VSMC Proliferation

The endothelium regulates the contractile status of vascular smooth muscle cells (VSMCs).1 The interaction between endothelial cells (ECs) and VSMCs plays an important role in regulating vascular homeostasis. ECs release vasoactive factors, such as prostacyclin, (nitric oxide [NO]), and endothelium-derived hyperpolarizing factor, which participate in the regulation of vascular tone.1 Endothelial dysfunction induces the increased expression of adhesion molecules for inflammatory cells. Accumulated inflammatory cells generate an oxidizing environment, which involves abundant reactive oxygen species, inflammatory cytokines/chemokines, and growth factors that contribute to VSMC phenotypic modulation.2 Thus, EC damage is a trigger underlying VSMC phenotypic change and pathological vascular remodeling. Article, see p 1120 Under normal physiological conditions, VSMCs in the media of vessels are quiescent with a low turnover rate and insignificant secretory activity. They are highly differentiated cells, which show a contractile phenotype and regulate vascular tone. However, VSMCs are among the most plastic of all cells in their ability to respond to different stimuli and retain a degree of plasticity to allow phenotypic modulation. It is thought that VSMCs change from a quiescent/contractile to an active/synthetic phenotype in several vascular diseases.3 Synthetic VSMCs downregulate contractile proteins and upregulate growth factors, receptors, extracellular matrix proteases, and inflammatory proteins. Besides VSMC phenotypic change, the transdifferentiation of adventitial fibroblasts,4 the differentiation of progenitor cells/stem cells,5,6 and endothelial-to-mesenchymal transition may also contribute as the sources of synthetic VSMCs.7 Regardless of the sources, synthetic VSMCs proliferate, migrate, and secrete proteins, including extracellular matrix proteases, proinflammatory cytokines/chemokines, and growth factors. Therefore, it is of great interest to develop novel strategies targeting the VSMC phenotypic change from the contractile to synthetic state. Vascular ECs themselves produce abundant …

Prognostic Impacts of Plasma Levels of Cyclophilin A in Patients With Coronary Artery Disease

Objective— Cyclophilin A (CyPA) is secreted from vascular smooth muscle cells, inflammatory cells, and activated platelets in response to oxidative stress. We have recently demonstrated that plasma CyPA level is a novel biomarker for diagnosing coronary artery disease. However, it remains to be elucidated whether plasma CyPA levels also have a prognostic impact in such patients. Approach and Results— In 511 consecutive patients undergoing diagnostic coronary angiography, we measured the plasma levels of CyPA, high-sensitivity C-reactive protein (hsCRP), and brain natriuretic peptide and evaluated their prognostic impacts during the follow-up (42 months, interquartile range: 25–55 months). Higher CyPA levels (≥12 ng/mL) were significantly associated with all-cause death, rehospitalization, and coronary revascularization. Higher hsCRP levels (≥1 mg/L) were also significantly correlated with the primary end point and all-cause death, but not with rehospitalization or coronary revascularization. Similarly, higher brain natriuretic peptide levels (≥100 pg/mL) were significantly associated with all-cause death and rehospitalization, but not with coronary revascularization. Importantly, the combination of CyPA (≥12 ng/mL) and hsCRP (≥1 mg/L) was more significantly associated with all-cause death (hazard ratio, 21.2; 95% confidence interval, 4.9–92.3,; P<0.001) than CyPA (≥12 ng/mL) or hsCRP (≥1 mg/L) alone. Conclusions— The results indicate that plasma CyPA levels can be used to predict all-cause death, rehospitalization, and coronary revascularization in patients with coronary artery disease and that when combined with other biomarkers (hsCRP and brain natriuretic peptide levels), the CyPA levels have further enhanced prognostic impacts in those patients.

SmgGDS as a Crucial Mediator of the Inhibitory Effects of Statins on Cardiac Hypertrophy and Fibrosis Novel Mechanism of the Pleiotropic Effects of Statins

The detailed molecular mechanisms of the pleiotropic effects of statins remain to be fully elucidated. Here, we hypothesized that cardioprotective effects of statins are mediated by small GTP-binding protein GDP dissociation stimulator (SmgGDS). SmgGDS+/– and wild-type (WT) mice were treated with continuous infusion of angiotensin II (Ang II) for 2 weeks with and without oral treatment with atorvastatin or pravastatin. At 2 weeks, the extents of Ang II–induced cardiac hypertrophy and fibrosis were comparable between the 2 genotypes. However, statins significantly attenuated cardiomyocyte hypertrophy and fibrosis in WT mice, but not in SmgGDS+/– mice. In SmgGDS+/– cardiac fibroblasts (CFs), Rac1 expression, extracellular signal–regulated kinases 1/2 activity, Rho-kinase activity, and inflammatory cytokines secretion in response to Ang II were significantly increased when compared with WT CFs. Atorvastatin significantly reduced Rac1 expression and oxidative stress in WT CFs, but not in SmgGDS+/– CFs. Furthermore, Bio-plex analysis revealed significant upregulations of inflammatory cytokines/chemokines and growth factors in SmgGDS+/– CFs when compared with WT CFs. Importantly, conditioned medium from SmgGDS+/– CFs increased B-type natriuretic peptide expression in rat cardiomyocytes to a greater extent than that from WT CFs. Furthermore, atorvastatin significantly increased SmgGDS secretion from mouse CFs. Finally, treatment with recombinant SmgGDS significantly reduced Rac1 expression in SmgGDS+/– CFs. These results indicate that both intracellular and extracellular SmgGDS play crucial roles in the inhibitory effects of statins on cardiac hypertrophy and fibrosis, partly through inhibition of Rac1, Rho kinase, and extracellular signal–regulated kinase 1/2 pathways, demonstrating the novel mechanism of the pleiotropic effects of statins.

Normal spectral emissivity measurement of molten copper using an electromagnetic levitator superimposed with a static magnetic field

The normal spectral emissivity of molten copper was determined in the wavelength range of 780?920?nm and in the temperature range of 1288?1678?K, by directly measuring the radiance emitted by an electromagnetically levitated molten copper droplet under a static magnetic field of 1.5 T. The spectrometer for radiance measurement was calibrated using the relation between the theoretical blackbody radiance from Plancks law and the light intensity of a quasi-blackbody radiation source measured using a spectrometer at a given temperature. As a result, the normal spectral emissivity of molten copper was determined as 0.075 ? 0.011 at a wavelength of 807?nm, and it was found that its temperature dependence is negligible in the entire measurement temperature range tested. In addition, the results of the normal spectral emissivity and its wavelength dependence were discussed, in comparison with those obtained using the Drude free-electron model.

Molecular mechanisms of the angiogenic effects of low-energy shock wave therapy: roles of mechanotransduction

We have previously demonstrated that low-energy extracorporeal cardiac shock wave (SW) therapy improves myocardial ischemia through enhanced myocardial angiogenesis in a porcine model of chronic myocardial ischemia and in patients with refractory angina pectoris. However, the detailed molecular mechanisms for the SW-induced angiogenesis remain unclear. In this study, we thus examined the effects of SW irradiation on intracellular signaling pathways in vitro. Cultured human umbilical vein endothelial cells (HUVECs) were treated with 800 shots of low-energy SW (1 Hz at an energy level of 0.03 mJ/mm(2)). The SW therapy significantly upregulated mRNA expression and protein levels of vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS). The SW therapy also enhanced phosphorylation of extracellular signal-regulated kinase 1/2 (Erk1/2) and Akt. Furthermore, the SW therapy enhanced phosphorylation of caveolin-1 and the expression of HUTS-4 that represents β1-integrin activity. These results suggest that caveolin-1 and β1-integrin are involved in the SW-induced activation of angiogenic signaling pathways. To further examine the signaling pathways involved in the SW-induced angiogenesis, HUVECs were transfected with siRNA of either β1-integrin or caveolin-1. Knockdown of either caveolin-1 or β1-integrin suppressed the SW-induced phosphorylation of Erk1/2 and Akt and upregulation of VEGF and eNOS. Knockdown of either caveolin-1 or β1-integrin also suppressed SW-induced enhancement of HUVEC migration in scratch assay. These results suggest that activation of mechanosensors on cell membranes, such as caveolin-1 and β1-integrin, and subsequent phosphorylation of Erk and Akt may play pivotal roles in the SW-induced angiogenesis.

Activated TAFI Promotes the Development of Chronic Thromboembolic Pulmonary Hypertension: A Possible Novel Therapeutic Target.

Rationale: Pulmonary hypertension is a fatal disease; however, its pathogenesis still remains to be elucidated. Thrombin-activatable fibrinolysis inhibitor (TAFI) is synthesized by the liver and inhibits fibrinolysis. Plasma TAFI levels are significantly increased in chronic thromboembolic pulmonary hypertension (CTEPH) patients. Objective: To determine the role of activated TAFI (TAFIa) in the development of CTEPH. Methods and Results: Immunostaining showed that TAFI and its binding partner thrombomodulin (TM) were highly expressed in the pulmonary arteries (PAs) and thrombus in patients with CTEPH. Moreover, plasma levels of TAFIa were increased 10-fold in CTEPH patients compared with controls. In mice, chronic hypoxia caused a 25-fold increase in plasma levels of TAFIa with increased plasma levels of thrombin and TM, which led to thrombus formation in PA, vascular remodeling, and pulmonary hypertension. Consistently, plasma clot lysis time was positively correlated with plasma TAFIa levels in mice. Additionally, overexpression of TAFIa caused organized thrombus with multiple obstruction of PA flow and reduced survival rate under hypoxia in mice. Bone marrow transplantation showed that circulating plasma TAFI from the liver, not in the bone marrow, was activated locally in PA endothelial cells through interactions with thrombin and TM. Mechanistic experiments demonstrated that TAFIa increased PA endothelial permeability, smooth muscle cell proliferation, and monocyte/macrophage activation. Importantly, TAFIa inhibitor and peroxisome proliferator–activated receptor-&agr; agonists significantly reduced TAFIa and ameliorated animal models of pulmonary hypertension in mice and rats. Conclusions: These results indicate that TAFIa could be a novel biomarker and realistic therapeutic target of CTEPH.