Hideto Sano - Academia.edu (original) (raw)

Papers by Hideto Sano

Research paper thumbnail of Activated platelet-based inhibition of fibrinolysis via thrombin-activatable fibrinolysis inhibitor activation system

Blood Advances, Nov 9, 2020

Activated platelets facilitate plasma TAFI activation in a solubleand/or platelet-derived thrombo... more Activated platelets facilitate plasma TAFI activation in a solubleand/or platelet-derived thrombomodulindependent manner. • TAFIa inhibitor enhances accumulation of plasminogen and its propagation from activated platelets to periphery along with fibrinolysis. Our previous real-time imaging studies directly demonstrated the spatiotemporal regulation of clot formation and lysis by activated platelets. In addition to their procoagulant functions, platelets enhanced profibrinolytic potential by augmenting the accumulation of tissue-type plasminogen activator (tPA) and plasminogen, in vivo in a murine microthrombus model, and in vitro in a platelet-containing plasma clot model. To clarify the role of thrombinactivatable fibrinolysis inhibitor (TAFI), which regulates coagulation-dependent antifibrinolytic potential, we analyzed tPA-induced clot lysis times in platelet-containing plasma. Platelets prolonged clot lysis times in a concentration-dependent manner, which were successfully abolished by a thrombomodulin-neutralizing antibody or an activated TAFI inhibitor (TAFIaI). The results obtained using TAFI-or factor XIII-deficient plasma suggested that TAFI in plasma, but not in platelets, was essential for this prolongation, though its crosslinkage with fibrin was not necessary. Confocal laser scanning microscopy revealed that fluorescence-labeled plasminogen accumulated on activated platelet surfaces and propagated to the periphery, similar to the propagation of fibrinolysis. Plasminogen accumulation and propagation were both enhanced by TAFIaI, but only accumulation was enhanced by thrombomodulin-neutralizing antibody. Labeled TAFI also accumulated on both fibrin fibers and activated platelet surfaces, which were Lys-binding-site-dependent and Lys-binding-site-independent, respectively. Finally, TAFIaI significantly prolonged the occlusion times of tPA-containing whole blood in a microchip-based flow chamber system, suggesting that TAFI attenuated the tPA-dependent prolongation of clot formation under flow. Thus, activated platelet surfaces are targeted by plasma TAFI, to attenuate plasminogen accumulation and fibrinolysis, which may contribute to thrombogenicity under flow.

Research paper thumbnail of Blockade of Platelet-Derived Growth Factor Receptor-β Pathway Induces Apoptosis of Vascular Endothelial Cells and Disrupts Glomerular Capillary Formation in Neonatal Mice

American Journal of Pathology, Jul 1, 2002

Platelet-derived growth factor (PDGF), a potent chemotactic and proliferation factor for mesenchy... more Platelet-derived growth factor (PDGF), a potent chemotactic and proliferation factor for mesenchymalderived cells, has been demonstrated to play critical roles in kidney development. Two receptors for PDGF, PDGFR-␣ and PDGFR-␤, have been identified and we previously analyzed the effects of blockade of PDGFR-␣ signal in neonatal mice. In the current study, we examined the role of PDGFR-␤ in glomerular development by blocking PDGFR-␤ signal in neonatal mice by administration of antagonistic anti-PDGFR-␤ monoclonal antibody. Unlike the mice injected with anti-PDGFR-␣ antibody, the mice injected daily with anti-PDGFR-␤ antibody could be kept alive at least for 2 weeks after birth but showed severe disruption of the glomerular structure, whereas no apparent deformation was observed in the collecting ducts. In the disrupted glomeruli, the number of the mesangial cells was reduced markedly. Electron microscopic analysis and immunohistochemical studies with terminal deoxynucleotidyl transferase nick-end labeling staining revealed that the capillary endothelial cells of the glomeruli in the outer cortex region underwent apoptosis. However, the glomeruli located near the medulla were less affected. Because PDGFR-␤ is not expressed in the endothelial cells, the effects of the blockade of PDGFR-␤ might have caused glomerular endothelial cell apoptosis by inducing the loss of mesangial cells and/or pericytes.

Research paper thumbnail of Interaction between macrophage and smooth muscle progenitor cells in vascular remodeling lesions in mice

Japanese Circulation Journal-english Edition, 2002

Research paper thumbnail of Roles of thromboxane A2 and prostacyclin in the development of atherosclerosis in apoE-deficient mice

Journal of Clinical Investigation, 2004

Production of thromboxane (TX) A 2 and PG I 2 /prostacyclin (PGI 2) is increased in patients with... more Production of thromboxane (TX) A 2 and PG I 2 /prostacyclin (PGI 2) is increased in patients with atherosclerosis. However, their roles in atherogenesis have not been critically defined. To examine this issue, we cross-bred atherosclerosis-prone apoE-deficient mice with mice deficient in either the TXA receptor (TP) or the PGI receptor (IP). Although they showed levels of serum cholesterol and triglyceride similar to those of apoE-deficient mice, apoE-/-TP-/mice exhibited a significant delay in atherogenesis, and apoE-/-IP-/mice exhibited a significant acceleration in atherogenesis compared with mice deficient in apoE alone. The plaques in apoE-/-IP-/mice showed partial endothelial disruption and exhibited enhanced expression of ICAM-1 and decreased expression of platelet endothelial cell adhesion molecule 1 (PECAM-1) in the overlying endothelial cells compared with those of apoE-/-TP-/mice. Platelet activation with thrombin ex vivo revealed higher and lower sensitivity for surface P-selectin expression in platelets of apoE-/-IP-/and apoE-/-TP-/mice, respectively, than in those of apoE-/mice. Intravital microscopy of the common carotid artery revealed a significantly greater number of leukocytes rolling on the vessel walls in apoE-/-IP-/mice than in either apoE-/-TP-/or apoE-/mice. We conclude that TXA 2 promotes and PGI 2 prevents the initiation and progression of atherogenesis through control of platelet activation and leukocyte-endothelial cell interaction.

Research paper thumbnail of Functional Blockade of Platelet-Derived Growth Factor Receptor-β but Not of Receptor-α Prevents Vascular Smooth Muscle Cell Accumulation in Fibrous Cap Lesions in Apolipoprotein E–Deficient Mice

Circulation, 2001

Background —The vascular smooth muscle cell (VSMC) is the central cell component involved in the ... more Background —The vascular smooth muscle cell (VSMC) is the central cell component involved in the fibroproliferative response in atherogenesis. As the lesion advances, VSMCs migrate from the media into the subendothelial space, thereby forming fibrous plaque lesions. Platelet-derived growth factor (PDGF) has been known to be a potent chemoattractant and mitogen for SMCs, but the pathophysiological role of the 2 PDGF receptors, receptor-α (PDGFR-α) and receptor-β (PDGFR-β) in atherogenesis is poorly understood. To clarify this problem, we prepared antagonistic rat monoclonal antibodies, APA5 and APB5, against murine PDGFR-α and PDGFR-β, respectively. Methods and Results —Apolipoprotein E–deficient mice were fed a high-fat diet containing 0.3% cholesterol from 6 weeks of age and subjected to injection with 1 mg/d IP of either antibody from 12 to 18 weeks every other day. In the mice injected with APB5, the aortic atherosclerotic lesion size and the number of intimal VSMCs were reduced ...

Research paper thumbnail of Negative Regulation of VEGF-Induced Vascular Leakage by Blockade of Angiotensin II Type 1 Receptor

Arteriosclerosis, Thrombosis, and Vascular Biology, 2006

Objective— Permeability of blood vessels is essential for tissue homeostasis. However, disorganiz... more Objective— Permeability of blood vessels is essential for tissue homeostasis. However, disorganized hyperpermeability leads to progression of diseases. Vascular endothelial growth factor-A (VEGF) is a key regulator for leakiness of blood vessels and it has been reported that VEGF-mediated hyperpermeability was suppressed by angiopoietin-1 (Ang1). We found that Angiotensin-converting enzyme (ACE) was downregulated in endothelial cells by Ang1. ACE converts angiotensin I to angiotensin II (AII). Here, we studied the relationship between VEGF and AII relative to vascular permeability. Methods and Results— We showed that VEGF-mediated vascular hyperpermeability was suppressed in mice given AII type 1 receptor (AT1R) blocker (ARB); the effect was also seen in AT1R-deficient mice. In this system, we found that ARB inhibited VEGF-induced gap formation. Furthermore, we ascertained that angioedema induced by overexpression of VEGF decreased noticeably in ARB-treated ischemic mice. Conclusion...

Research paper thumbnail of Role of Bone Marrow–Derived Progenitor Cells in Cuff-Induced Vascular Injury in Mice

Arteriosclerosis, Thrombosis, and Vascular Biology, 2004

Objectives— Arterial injury results in vascular remodeling associated with proliferation and migr... more Objectives— Arterial injury results in vascular remodeling associated with proliferation and migration of smooth muscle cells (SMCs) and the development of intimal hyperplasia, which is a critical component of restenosis after angioplasty of human coronary arteries and an important feature of atherosclerotic lesions. However, the origin of SMCs and other cells in the development of vascular remodeling is not yet fully understood. Methods and Results— We utilized a cuff-induced vascular injury model after transplantation of the bone marrow (BM) from green fluorescent protein (GFP)-transgenic mice. We found that macrophages were major cells recruited to the adventitia of the vascular injury lesion along with SMCs and endothelial cells (ECs). While investigating whether those cells are derived from the donor, we found that most of the macrophages were GFP-positive, and some of the SMCs and ECs were also GFP-positive. Administration of the anti–c- fms antibody resulted in a marked decre...

Research paper thumbnail of アポリポプロテイン E ケッソン マウス ニ オイテ ケッショウバン ユライ ゾウショク インシ アルファ ジュヨウタイ デワ ナク ベータ ジュヨウタイ ノ キノウテキ シャダン ガ ケッカン ヘイカツキン サイボウ ノ センイセイ コウハン リョウイキ エノ チクセキ オ ヨクセイ スル

Kyoto University (京都大学)0048新制・課程博士博士(医学)甲第9093号医博第2401号新制||医||778(附属図書館)UT51-2001-G813京都大学大学院医学研究... more Kyoto University (京都大学)0048新制・課程博士博士(医学)甲第9093号医博第2401号新制||医||778(附属図書館)UT51-2001-G813京都大学大学院医学研究科内科系専攻(主査)教授 内山 卓, 教授 中尾 一和, 教授 北 徹学位規則第4条第1項該

Research paper thumbnail of RESEARCH ARTICLE Endogenously Generated Plasmin at the Vascular Wall Injury Site Amplifies Lysine Binding Site-Dependent Plasminogen Accumulation in Microthrombi

The fibrinolytic system plays a pivotal role in the regulation of hemostasis; however, it remains... more The fibrinolytic system plays a pivotal role in the regulation of hemostasis; however, it remains unclear how and when the system is triggered to induce thrombolysis. Using intra-vital confocal fluorescence microscopy, we investigated the process of plasminogen bind-ing to laser-induced platelet-rich microthrombi generated in the mesenteric vein of transgen-ic mice expressing green fluorescent protein (GFP). The accumulation of GFP-expressing platelets as well as exogenously infused Alexa Fluor 568-labeled Glu-plasminogen (Glu-plg) on the injured vessel wall was assessed by measuring the increase in the correspond-ing fluorescence intensities. Glu-plg accumulated in a time-dependent manner in the center of the microthrombus, where phosphatidylserine is exposed on platelet surfaces and fibrin formation takes place. The rates of binding of Glu-plg in the presence of ε-aminocaproic acid and carboxypeptidase B, as well as the rates of binding of mini-plasminogen lacking kringle domains ...

Research paper thumbnail of Pre-administration of a carboxypeptidase inhibitor enhances tPA-induced thrombolysis in mouse microthrombi: Evidence from intravital imaging analysis

Thrombosis Research, 2022

Thrombolysis using recombinant tissue-type plasminogen activator (rt-PA) is the pharmacological t... more Thrombolysis using recombinant tissue-type plasminogen activator (rt-PA) is the pharmacological treatment of choice in acute thrombotic events. However, a narrow therapeutic window and bleeding complications limit its use. We describe the role of carboxypeptidase inhibitor from potato tuber (PTCI), an inhibitor of activated thrombin-activatable fibrinolysis inhibitor (TAFIa), on Glu-plasminogen accumulation and microthrombus dynamics in vivo and demonstrate its influence on rt-PA-mediated thrombolysis. Materials and methods: In conjunction with real-time intravital two-photon excitation fluorescence microscopy, we produced and imaged laser-induced microthrombi in the mesenteric venules of Green Fluorescent Protein (GFP)-expressing mice. We examined microthrombus dynamics and thrombolysis patterns in vivo by measuring the changes in the fluorescence intensity of labeled Glu-plasminogen following administration of epsilon aminocaproic acid (EACA), PTCI, and rt-PA. Results: PTCI enhanced Glu-plasminogen accumulation at the core of the thrombus by inhibiting TAFIa, while EACA inhibited this process. Exogenous rt-PA effectively triggered Glu-plasminogen activation within the thrombus and promoted thrombolysis. Administration of PTCI and rt-PA together showed no significant benefit on thrombolysis compared to rt-PA administration alone. However, early-phase systemic administration of PTCI before thrombolytic therapy by rt-PA expedited clot lysis as evidenced by significantly faster time to reach peak Glu-plasminogen fluorescence intensity and shorter time to achieve near-complete clot lysis (P = 0.014 and P = 0.003, respectively). Conclusions: PTCI potentiates rt-PA-mediated thrombolysis when administered early in acute thrombotic events. Further studies are warranted to explore the potential of TAFI inhibitors as adjunct agents in thrombolysis or thromboprophylaxis.

Research paper thumbnail of Analysis of Blood Vessel Formation and Self-Renewal of Hematopoietic Stem Cells

Research paper thumbnail of OE-075 Immunoglobulin Suppresses Experimental Atherosclerosis via the Fc Portion Independent of Hypercholesterolemia(Atherosclerosis, basic-1, The 71st Annual Scientific Meeting of the Japanese Circulation Society)

Research paper thumbnail of Febuxostat Pretreatment Attenuates Experimental Aortic Dissection in Mice

European Journal of Vascular and Endovascular Surgery, 2019

The dialytic performance was determined by the pre-and post-operative comparison of dialytic veno... more The dialytic performance was determined by the pre-and post-operative comparison of dialytic venous pressures (DVP) and dialytic flow (DF). The venography was used to confirm the clinical suspect of obstrucion. A predilatation by the use of high-pressure balloon was performed in the obstructions and a pre and post-stenting PTA was performed in all cases. Kaplan-Meier life-table analysis for the treated veins and AVF patency and Cox proportional hazards for time-dependent variables analysis were performed. Results-Over a total of 1613 patients submitted to vascular procedures related to AVF, EVT was performed in 15 (1%) CV cases: 12 (80%) stenoses and 3 (20%) obstructions. Two (13%) patients had a prosthetic and 13 (87%) a proximal AVF. Nine angioplasties, 4 stenting and 2 covered stents were performed in 5 brachiocephalic, 1 subclavian and 9 axillary segments. Technical success was 100% with no complications such as CV rupture or thrombosis. DVP was significantly reduced and DF significantly increased after the procedure (191AE15 mmHg vs 149AE14 mmHg; P¼.001 and 282AE19 ml/min vs 313AE22 ml/min; P.001, respectively). At a mean follow-up of 16 months, the primary patency of the treated veins was 67% and the AVF patency was 100%. Conclusion-EVT is a safe and effective treatment for CV obstruction and stenosis and allows to ameliorate AVF dialytic performance in terms of DVP and DF.

Research paper thumbnail of Roles of the fibrinolytic system in vascular hemostasis —Insights from the TAFI’s function

Japanese Journal of Thrombosis and Hemostasis, 2020

Research paper thumbnail of Ectopic Expression of PCSK9 by Smooth Muscle Cells Contributes to Aortic Dissection

Annals of vascular surgery, 2018

Acute aortic dissection (AAD) is a common disease among the elderly. Although several risk factor... more Acute aortic dissection (AAD) is a common disease among the elderly. Although several risk factors of AAD have been reported, the molecular mechanism underlying AAD development remains to be elucidated. Proprotein convertase subtilisin/kexin type 9 (PCSK9) increases low-density lipoprotein cholesterol levels in blood by preventing its clearance. Therefore, PCSK9 inhibition is a promising therapeutic approach to treat cardiovascular diseases (CVDs). The objective of this study was to elucidate the role of PCSK9 in the pathogenesis of AAD. We used fluorescence immunohistochemistry to assess PCSK9 expression in aortic tissues resected from 10 AAD patients and in the normal aorta from 5 autopsy samples as well as in spontaneously hyperlipidemic apolipoprotein E-deficient mice used as an experimental AD model. We revealed a characteristic distribution pattern of PCSK9 in atherosclerotic plaques and the degenerated tunica media in AAD tissues, which was rarely observed in normal aortic ti...

Research paper thumbnail of Hypoperfusion of the aortic wall secondary to degeneration of adventitial vasa vasorum causes abdominal aortic aneurysms

Current drug targets, Jan 22, 2018

An abdominal aortic aneurysm (AAA), which affects approximately 10% of Japanese men aged ≥ 65 yea... more An abdominal aortic aneurysm (AAA), which affects approximately 10% of Japanese men aged ≥ 65 years, is frequently associated with hypertension, dyslipidemia, and other lifestyle-related diseases. The development of an AAA is attributed to chronic inflammation concomitant with arteriosclerotic changes. However, an accurate pathomechanism associated with AAA remains uncertain, and questions such as why only a particular group/percentage of patients with arteriosclerosis develop aneurysms and how diabetes suppresses aneurysm development remain unanswered. We examined a novel mechanism to develop AAA based on histopathological findings following analysis of the human AAA tissues. Additionally, based on these findings, we developed a new animal model of AAA, in which the histopathological characteristics are similar to human AAA tissue. Recently, we identified stenosis of the vasa vasorum (VV) in aortic segments showing dilatation. The aorta is the largest artery in our circulatory syst...

Research paper thumbnail of Bidirectional functions of thrombin on fibrinolysis: Evidence of thrombin-dependent enhancement of fibrinolysis provided by spontaneous plasma clot lysis

Thrombosis research, 2016

Besides procoagulant activity, thrombin exhibits anticoagulant and profibrinolytic activities. We... more Besides procoagulant activity, thrombin exhibits anticoagulant and profibrinolytic activities. We demonstrated that the euglobulin clot lysis time (ECLT) was shortened by endogenously generated thrombin as a result of the inactivation of plasminogen activator inhibitor type 1 (PAI-1). In contrast, thrombin suppressed fibrinolytic activity through the activation of thrombin activatable fibrinolysis inhibitor (TAFI). Here, using three different clot lysis assays of the ECLT, the tissue plasminogen activator supplemented plasma clot lysis time (tPA-PCLT) and the spontaneous plasma clot lysis time (s-PCLT), we analyzed how the coagulation process modifies fibrinolysis. The ECLT was shortened by exogenously supplemented thrombin in a dose-dependent manner in the absence of calcium ion (Ca(++)), whereas this shortening was not observed in the presence of Ca(++) where endogenous prothrombin was effectively activated to thrombin. This shortening was also not observed for the tPA-PCLT, in wh...

Research paper thumbnail of Activities of wild-type and variant tissue-type plasminogen activators retained on vascular endothelial cells

Research paper thumbnail of Endogenously generated plasmin at the vascular wall injury site amplifies lysine binding site-dependent plasminogen accumulation in microthrombi

PloS one, 2015

The fibrinolytic system plays a pivotal role in the regulation of hemostasis; however, it remains... more The fibrinolytic system plays a pivotal role in the regulation of hemostasis; however, it remains unclear how and when the system is triggered to induce thrombolysis. Using intra-vital confocal fluorescence microscopy, we investigated the process of plasminogen binding to laser-induced platelet-rich microthrombi generated in the mesenteric vein of transgenic mice expressing green fluorescent protein (GFP). The accumulation of GFP-expressing platelets as well as exogenously infused Alexa Fluor 568-labeled Glu-plasminogen (Glu-plg) on the injured vessel wall was assessed by measuring the increase in the corresponding fluorescence intensities. Glu-plg accumulated in a time-dependent manner in the center of the microthrombus, where phosphatidylserine is exposed on platelet surfaces and fibrin formation takes place. The rates of binding of Glu-plg in the presence of ε-aminocaproic acid and carboxypeptidase B, as well as the rates of binding of mini-plasminogen lacking kringle domains 1-4...

Research paper thumbnail of Binding of Thrombin-Activated Platelets to a Fibrin Scaffold through αIIbβ3 Evokes Phosphatidylserine Exposure on Their Cell Surface

PLoS ONE, 2013

Recently, by employing intra-vital confocal microscopy, we demonstrated that platelets expose pho... more Recently, by employing intra-vital confocal microscopy, we demonstrated that platelets expose phosphatidylserine (PS) and fibrin accumulate only in the center of the thrombus but not in its periphery. To address the question how exposure of platelet anionic phospholipids is regulated within the thrombus, an in-vitro experiment using diluted platelet-rich plasma was employed, in which the fibrin network was formed in the presence of platelets, and PS exposure on the platelet surface was analyzed using Confocal Laser Scanning Microscopy. Almost all platelets exposed PS after treatment with tissue factor, thrombin or ionomycin. Argatroban abrogated fibrin network formation in all samples, however, platelet PS exposure was inhibited only in tissue factor-and thrombin-treated samples but not in ionomycin-treated samples. FK633, an a IIb b 3 antagonist, and cytochalasin B impaired platelet binding to the fibrin scaffold and significantly reduced PS exposure evoked by thrombin. Gly-Pro-Arg-Pro amide abrogated not only fibrin network formation, but also PS exposure on platelets without suppressing platelet binding to fibrin/fibrinogen. These results suggest that outside-in signals in platelets generated by their binding to the rigid fibrin network are essential for PS exposure after thrombin treatment.

Research paper thumbnail of Activated platelet-based inhibition of fibrinolysis via thrombin-activatable fibrinolysis inhibitor activation system

Blood Advances, Nov 9, 2020

Activated platelets facilitate plasma TAFI activation in a solubleand/or platelet-derived thrombo... more Activated platelets facilitate plasma TAFI activation in a solubleand/or platelet-derived thrombomodulindependent manner. • TAFIa inhibitor enhances accumulation of plasminogen and its propagation from activated platelets to periphery along with fibrinolysis. Our previous real-time imaging studies directly demonstrated the spatiotemporal regulation of clot formation and lysis by activated platelets. In addition to their procoagulant functions, platelets enhanced profibrinolytic potential by augmenting the accumulation of tissue-type plasminogen activator (tPA) and plasminogen, in vivo in a murine microthrombus model, and in vitro in a platelet-containing plasma clot model. To clarify the role of thrombinactivatable fibrinolysis inhibitor (TAFI), which regulates coagulation-dependent antifibrinolytic potential, we analyzed tPA-induced clot lysis times in platelet-containing plasma. Platelets prolonged clot lysis times in a concentration-dependent manner, which were successfully abolished by a thrombomodulin-neutralizing antibody or an activated TAFI inhibitor (TAFIaI). The results obtained using TAFI-or factor XIII-deficient plasma suggested that TAFI in plasma, but not in platelets, was essential for this prolongation, though its crosslinkage with fibrin was not necessary. Confocal laser scanning microscopy revealed that fluorescence-labeled plasminogen accumulated on activated platelet surfaces and propagated to the periphery, similar to the propagation of fibrinolysis. Plasminogen accumulation and propagation were both enhanced by TAFIaI, but only accumulation was enhanced by thrombomodulin-neutralizing antibody. Labeled TAFI also accumulated on both fibrin fibers and activated platelet surfaces, which were Lys-binding-site-dependent and Lys-binding-site-independent, respectively. Finally, TAFIaI significantly prolonged the occlusion times of tPA-containing whole blood in a microchip-based flow chamber system, suggesting that TAFI attenuated the tPA-dependent prolongation of clot formation under flow. Thus, activated platelet surfaces are targeted by plasma TAFI, to attenuate plasminogen accumulation and fibrinolysis, which may contribute to thrombogenicity under flow.

Research paper thumbnail of Blockade of Platelet-Derived Growth Factor Receptor-β Pathway Induces Apoptosis of Vascular Endothelial Cells and Disrupts Glomerular Capillary Formation in Neonatal Mice

American Journal of Pathology, Jul 1, 2002

Platelet-derived growth factor (PDGF), a potent chemotactic and proliferation factor for mesenchy... more Platelet-derived growth factor (PDGF), a potent chemotactic and proliferation factor for mesenchymalderived cells, has been demonstrated to play critical roles in kidney development. Two receptors for PDGF, PDGFR-␣ and PDGFR-␤, have been identified and we previously analyzed the effects of blockade of PDGFR-␣ signal in neonatal mice. In the current study, we examined the role of PDGFR-␤ in glomerular development by blocking PDGFR-␤ signal in neonatal mice by administration of antagonistic anti-PDGFR-␤ monoclonal antibody. Unlike the mice injected with anti-PDGFR-␣ antibody, the mice injected daily with anti-PDGFR-␤ antibody could be kept alive at least for 2 weeks after birth but showed severe disruption of the glomerular structure, whereas no apparent deformation was observed in the collecting ducts. In the disrupted glomeruli, the number of the mesangial cells was reduced markedly. Electron microscopic analysis and immunohistochemical studies with terminal deoxynucleotidyl transferase nick-end labeling staining revealed that the capillary endothelial cells of the glomeruli in the outer cortex region underwent apoptosis. However, the glomeruli located near the medulla were less affected. Because PDGFR-␤ is not expressed in the endothelial cells, the effects of the blockade of PDGFR-␤ might have caused glomerular endothelial cell apoptosis by inducing the loss of mesangial cells and/or pericytes.

Research paper thumbnail of Interaction between macrophage and smooth muscle progenitor cells in vascular remodeling lesions in mice

Japanese Circulation Journal-english Edition, 2002

Research paper thumbnail of Roles of thromboxane A2 and prostacyclin in the development of atherosclerosis in apoE-deficient mice

Journal of Clinical Investigation, 2004

Production of thromboxane (TX) A 2 and PG I 2 /prostacyclin (PGI 2) is increased in patients with... more Production of thromboxane (TX) A 2 and PG I 2 /prostacyclin (PGI 2) is increased in patients with atherosclerosis. However, their roles in atherogenesis have not been critically defined. To examine this issue, we cross-bred atherosclerosis-prone apoE-deficient mice with mice deficient in either the TXA receptor (TP) or the PGI receptor (IP). Although they showed levels of serum cholesterol and triglyceride similar to those of apoE-deficient mice, apoE-/-TP-/mice exhibited a significant delay in atherogenesis, and apoE-/-IP-/mice exhibited a significant acceleration in atherogenesis compared with mice deficient in apoE alone. The plaques in apoE-/-IP-/mice showed partial endothelial disruption and exhibited enhanced expression of ICAM-1 and decreased expression of platelet endothelial cell adhesion molecule 1 (PECAM-1) in the overlying endothelial cells compared with those of apoE-/-TP-/mice. Platelet activation with thrombin ex vivo revealed higher and lower sensitivity for surface P-selectin expression in platelets of apoE-/-IP-/and apoE-/-TP-/mice, respectively, than in those of apoE-/mice. Intravital microscopy of the common carotid artery revealed a significantly greater number of leukocytes rolling on the vessel walls in apoE-/-IP-/mice than in either apoE-/-TP-/or apoE-/mice. We conclude that TXA 2 promotes and PGI 2 prevents the initiation and progression of atherogenesis through control of platelet activation and leukocyte-endothelial cell interaction.

Research paper thumbnail of Functional Blockade of Platelet-Derived Growth Factor Receptor-β but Not of Receptor-α Prevents Vascular Smooth Muscle Cell Accumulation in Fibrous Cap Lesions in Apolipoprotein E–Deficient Mice

Circulation, 2001

Background —The vascular smooth muscle cell (VSMC) is the central cell component involved in the ... more Background —The vascular smooth muscle cell (VSMC) is the central cell component involved in the fibroproliferative response in atherogenesis. As the lesion advances, VSMCs migrate from the media into the subendothelial space, thereby forming fibrous plaque lesions. Platelet-derived growth factor (PDGF) has been known to be a potent chemoattractant and mitogen for SMCs, but the pathophysiological role of the 2 PDGF receptors, receptor-α (PDGFR-α) and receptor-β (PDGFR-β) in atherogenesis is poorly understood. To clarify this problem, we prepared antagonistic rat monoclonal antibodies, APA5 and APB5, against murine PDGFR-α and PDGFR-β, respectively. Methods and Results —Apolipoprotein E–deficient mice were fed a high-fat diet containing 0.3% cholesterol from 6 weeks of age and subjected to injection with 1 mg/d IP of either antibody from 12 to 18 weeks every other day. In the mice injected with APB5, the aortic atherosclerotic lesion size and the number of intimal VSMCs were reduced ...

Research paper thumbnail of Negative Regulation of VEGF-Induced Vascular Leakage by Blockade of Angiotensin II Type 1 Receptor

Arteriosclerosis, Thrombosis, and Vascular Biology, 2006

Objective— Permeability of blood vessels is essential for tissue homeostasis. However, disorganiz... more Objective— Permeability of blood vessels is essential for tissue homeostasis. However, disorganized hyperpermeability leads to progression of diseases. Vascular endothelial growth factor-A (VEGF) is a key regulator for leakiness of blood vessels and it has been reported that VEGF-mediated hyperpermeability was suppressed by angiopoietin-1 (Ang1). We found that Angiotensin-converting enzyme (ACE) was downregulated in endothelial cells by Ang1. ACE converts angiotensin I to angiotensin II (AII). Here, we studied the relationship between VEGF and AII relative to vascular permeability. Methods and Results— We showed that VEGF-mediated vascular hyperpermeability was suppressed in mice given AII type 1 receptor (AT1R) blocker (ARB); the effect was also seen in AT1R-deficient mice. In this system, we found that ARB inhibited VEGF-induced gap formation. Furthermore, we ascertained that angioedema induced by overexpression of VEGF decreased noticeably in ARB-treated ischemic mice. Conclusion...

Research paper thumbnail of Role of Bone Marrow–Derived Progenitor Cells in Cuff-Induced Vascular Injury in Mice

Arteriosclerosis, Thrombosis, and Vascular Biology, 2004

Objectives— Arterial injury results in vascular remodeling associated with proliferation and migr... more Objectives— Arterial injury results in vascular remodeling associated with proliferation and migration of smooth muscle cells (SMCs) and the development of intimal hyperplasia, which is a critical component of restenosis after angioplasty of human coronary arteries and an important feature of atherosclerotic lesions. However, the origin of SMCs and other cells in the development of vascular remodeling is not yet fully understood. Methods and Results— We utilized a cuff-induced vascular injury model after transplantation of the bone marrow (BM) from green fluorescent protein (GFP)-transgenic mice. We found that macrophages were major cells recruited to the adventitia of the vascular injury lesion along with SMCs and endothelial cells (ECs). While investigating whether those cells are derived from the donor, we found that most of the macrophages were GFP-positive, and some of the SMCs and ECs were also GFP-positive. Administration of the anti–c- fms antibody resulted in a marked decre...

Research paper thumbnail of アポリポプロテイン E ケッソン マウス ニ オイテ ケッショウバン ユライ ゾウショク インシ アルファ ジュヨウタイ デワ ナク ベータ ジュヨウタイ ノ キノウテキ シャダン ガ ケッカン ヘイカツキン サイボウ ノ センイセイ コウハン リョウイキ エノ チクセキ オ ヨクセイ スル

Kyoto University (京都大学)0048新制・課程博士博士(医学)甲第9093号医博第2401号新制||医||778(附属図書館)UT51-2001-G813京都大学大学院医学研究... more Kyoto University (京都大学)0048新制・課程博士博士(医学)甲第9093号医博第2401号新制||医||778(附属図書館)UT51-2001-G813京都大学大学院医学研究科内科系専攻(主査)教授 内山 卓, 教授 中尾 一和, 教授 北 徹学位規則第4条第1項該

Research paper thumbnail of RESEARCH ARTICLE Endogenously Generated Plasmin at the Vascular Wall Injury Site Amplifies Lysine Binding Site-Dependent Plasminogen Accumulation in Microthrombi

The fibrinolytic system plays a pivotal role in the regulation of hemostasis; however, it remains... more The fibrinolytic system plays a pivotal role in the regulation of hemostasis; however, it remains unclear how and when the system is triggered to induce thrombolysis. Using intra-vital confocal fluorescence microscopy, we investigated the process of plasminogen bind-ing to laser-induced platelet-rich microthrombi generated in the mesenteric vein of transgen-ic mice expressing green fluorescent protein (GFP). The accumulation of GFP-expressing platelets as well as exogenously infused Alexa Fluor 568-labeled Glu-plasminogen (Glu-plg) on the injured vessel wall was assessed by measuring the increase in the correspond-ing fluorescence intensities. Glu-plg accumulated in a time-dependent manner in the center of the microthrombus, where phosphatidylserine is exposed on platelet surfaces and fibrin formation takes place. The rates of binding of Glu-plg in the presence of ε-aminocaproic acid and carboxypeptidase B, as well as the rates of binding of mini-plasminogen lacking kringle domains ...

Research paper thumbnail of Pre-administration of a carboxypeptidase inhibitor enhances tPA-induced thrombolysis in mouse microthrombi: Evidence from intravital imaging analysis

Thrombosis Research, 2022

Thrombolysis using recombinant tissue-type plasminogen activator (rt-PA) is the pharmacological t... more Thrombolysis using recombinant tissue-type plasminogen activator (rt-PA) is the pharmacological treatment of choice in acute thrombotic events. However, a narrow therapeutic window and bleeding complications limit its use. We describe the role of carboxypeptidase inhibitor from potato tuber (PTCI), an inhibitor of activated thrombin-activatable fibrinolysis inhibitor (TAFIa), on Glu-plasminogen accumulation and microthrombus dynamics in vivo and demonstrate its influence on rt-PA-mediated thrombolysis. Materials and methods: In conjunction with real-time intravital two-photon excitation fluorescence microscopy, we produced and imaged laser-induced microthrombi in the mesenteric venules of Green Fluorescent Protein (GFP)-expressing mice. We examined microthrombus dynamics and thrombolysis patterns in vivo by measuring the changes in the fluorescence intensity of labeled Glu-plasminogen following administration of epsilon aminocaproic acid (EACA), PTCI, and rt-PA. Results: PTCI enhanced Glu-plasminogen accumulation at the core of the thrombus by inhibiting TAFIa, while EACA inhibited this process. Exogenous rt-PA effectively triggered Glu-plasminogen activation within the thrombus and promoted thrombolysis. Administration of PTCI and rt-PA together showed no significant benefit on thrombolysis compared to rt-PA administration alone. However, early-phase systemic administration of PTCI before thrombolytic therapy by rt-PA expedited clot lysis as evidenced by significantly faster time to reach peak Glu-plasminogen fluorescence intensity and shorter time to achieve near-complete clot lysis (P = 0.014 and P = 0.003, respectively). Conclusions: PTCI potentiates rt-PA-mediated thrombolysis when administered early in acute thrombotic events. Further studies are warranted to explore the potential of TAFI inhibitors as adjunct agents in thrombolysis or thromboprophylaxis.

Research paper thumbnail of Analysis of Blood Vessel Formation and Self-Renewal of Hematopoietic Stem Cells

Research paper thumbnail of OE-075 Immunoglobulin Suppresses Experimental Atherosclerosis via the Fc Portion Independent of Hypercholesterolemia(Atherosclerosis, basic-1, The 71st Annual Scientific Meeting of the Japanese Circulation Society)

Research paper thumbnail of Febuxostat Pretreatment Attenuates Experimental Aortic Dissection in Mice

European Journal of Vascular and Endovascular Surgery, 2019

The dialytic performance was determined by the pre-and post-operative comparison of dialytic veno... more The dialytic performance was determined by the pre-and post-operative comparison of dialytic venous pressures (DVP) and dialytic flow (DF). The venography was used to confirm the clinical suspect of obstrucion. A predilatation by the use of high-pressure balloon was performed in the obstructions and a pre and post-stenting PTA was performed in all cases. Kaplan-Meier life-table analysis for the treated veins and AVF patency and Cox proportional hazards for time-dependent variables analysis were performed. Results-Over a total of 1613 patients submitted to vascular procedures related to AVF, EVT was performed in 15 (1%) CV cases: 12 (80%) stenoses and 3 (20%) obstructions. Two (13%) patients had a prosthetic and 13 (87%) a proximal AVF. Nine angioplasties, 4 stenting and 2 covered stents were performed in 5 brachiocephalic, 1 subclavian and 9 axillary segments. Technical success was 100% with no complications such as CV rupture or thrombosis. DVP was significantly reduced and DF significantly increased after the procedure (191AE15 mmHg vs 149AE14 mmHg; P¼.001 and 282AE19 ml/min vs 313AE22 ml/min; P.001, respectively). At a mean follow-up of 16 months, the primary patency of the treated veins was 67% and the AVF patency was 100%. Conclusion-EVT is a safe and effective treatment for CV obstruction and stenosis and allows to ameliorate AVF dialytic performance in terms of DVP and DF.

Research paper thumbnail of Roles of the fibrinolytic system in vascular hemostasis —Insights from the TAFI’s function

Japanese Journal of Thrombosis and Hemostasis, 2020

Research paper thumbnail of Ectopic Expression of PCSK9 by Smooth Muscle Cells Contributes to Aortic Dissection

Annals of vascular surgery, 2018

Acute aortic dissection (AAD) is a common disease among the elderly. Although several risk factor... more Acute aortic dissection (AAD) is a common disease among the elderly. Although several risk factors of AAD have been reported, the molecular mechanism underlying AAD development remains to be elucidated. Proprotein convertase subtilisin/kexin type 9 (PCSK9) increases low-density lipoprotein cholesterol levels in blood by preventing its clearance. Therefore, PCSK9 inhibition is a promising therapeutic approach to treat cardiovascular diseases (CVDs). The objective of this study was to elucidate the role of PCSK9 in the pathogenesis of AAD. We used fluorescence immunohistochemistry to assess PCSK9 expression in aortic tissues resected from 10 AAD patients and in the normal aorta from 5 autopsy samples as well as in spontaneously hyperlipidemic apolipoprotein E-deficient mice used as an experimental AD model. We revealed a characteristic distribution pattern of PCSK9 in atherosclerotic plaques and the degenerated tunica media in AAD tissues, which was rarely observed in normal aortic ti...

Research paper thumbnail of Hypoperfusion of the aortic wall secondary to degeneration of adventitial vasa vasorum causes abdominal aortic aneurysms

Current drug targets, Jan 22, 2018

An abdominal aortic aneurysm (AAA), which affects approximately 10% of Japanese men aged ≥ 65 yea... more An abdominal aortic aneurysm (AAA), which affects approximately 10% of Japanese men aged ≥ 65 years, is frequently associated with hypertension, dyslipidemia, and other lifestyle-related diseases. The development of an AAA is attributed to chronic inflammation concomitant with arteriosclerotic changes. However, an accurate pathomechanism associated with AAA remains uncertain, and questions such as why only a particular group/percentage of patients with arteriosclerosis develop aneurysms and how diabetes suppresses aneurysm development remain unanswered. We examined a novel mechanism to develop AAA based on histopathological findings following analysis of the human AAA tissues. Additionally, based on these findings, we developed a new animal model of AAA, in which the histopathological characteristics are similar to human AAA tissue. Recently, we identified stenosis of the vasa vasorum (VV) in aortic segments showing dilatation. The aorta is the largest artery in our circulatory syst...

Research paper thumbnail of Bidirectional functions of thrombin on fibrinolysis: Evidence of thrombin-dependent enhancement of fibrinolysis provided by spontaneous plasma clot lysis

Thrombosis research, 2016

Besides procoagulant activity, thrombin exhibits anticoagulant and profibrinolytic activities. We... more Besides procoagulant activity, thrombin exhibits anticoagulant and profibrinolytic activities. We demonstrated that the euglobulin clot lysis time (ECLT) was shortened by endogenously generated thrombin as a result of the inactivation of plasminogen activator inhibitor type 1 (PAI-1). In contrast, thrombin suppressed fibrinolytic activity through the activation of thrombin activatable fibrinolysis inhibitor (TAFI). Here, using three different clot lysis assays of the ECLT, the tissue plasminogen activator supplemented plasma clot lysis time (tPA-PCLT) and the spontaneous plasma clot lysis time (s-PCLT), we analyzed how the coagulation process modifies fibrinolysis. The ECLT was shortened by exogenously supplemented thrombin in a dose-dependent manner in the absence of calcium ion (Ca(++)), whereas this shortening was not observed in the presence of Ca(++) where endogenous prothrombin was effectively activated to thrombin. This shortening was also not observed for the tPA-PCLT, in wh...

Research paper thumbnail of Activities of wild-type and variant tissue-type plasminogen activators retained on vascular endothelial cells

Research paper thumbnail of Endogenously generated plasmin at the vascular wall injury site amplifies lysine binding site-dependent plasminogen accumulation in microthrombi

PloS one, 2015

The fibrinolytic system plays a pivotal role in the regulation of hemostasis; however, it remains... more The fibrinolytic system plays a pivotal role in the regulation of hemostasis; however, it remains unclear how and when the system is triggered to induce thrombolysis. Using intra-vital confocal fluorescence microscopy, we investigated the process of plasminogen binding to laser-induced platelet-rich microthrombi generated in the mesenteric vein of transgenic mice expressing green fluorescent protein (GFP). The accumulation of GFP-expressing platelets as well as exogenously infused Alexa Fluor 568-labeled Glu-plasminogen (Glu-plg) on the injured vessel wall was assessed by measuring the increase in the corresponding fluorescence intensities. Glu-plg accumulated in a time-dependent manner in the center of the microthrombus, where phosphatidylserine is exposed on platelet surfaces and fibrin formation takes place. The rates of binding of Glu-plg in the presence of ε-aminocaproic acid and carboxypeptidase B, as well as the rates of binding of mini-plasminogen lacking kringle domains 1-4...

Research paper thumbnail of Binding of Thrombin-Activated Platelets to a Fibrin Scaffold through αIIbβ3 Evokes Phosphatidylserine Exposure on Their Cell Surface

PLoS ONE, 2013

Recently, by employing intra-vital confocal microscopy, we demonstrated that platelets expose pho... more Recently, by employing intra-vital confocal microscopy, we demonstrated that platelets expose phosphatidylserine (PS) and fibrin accumulate only in the center of the thrombus but not in its periphery. To address the question how exposure of platelet anionic phospholipids is regulated within the thrombus, an in-vitro experiment using diluted platelet-rich plasma was employed, in which the fibrin network was formed in the presence of platelets, and PS exposure on the platelet surface was analyzed using Confocal Laser Scanning Microscopy. Almost all platelets exposed PS after treatment with tissue factor, thrombin or ionomycin. Argatroban abrogated fibrin network formation in all samples, however, platelet PS exposure was inhibited only in tissue factor-and thrombin-treated samples but not in ionomycin-treated samples. FK633, an a IIb b 3 antagonist, and cytochalasin B impaired platelet binding to the fibrin scaffold and significantly reduced PS exposure evoked by thrombin. Gly-Pro-Arg-Pro amide abrogated not only fibrin network formation, but also PS exposure on platelets without suppressing platelet binding to fibrin/fibrinogen. These results suggest that outside-in signals in platelets generated by their binding to the rigid fibrin network are essential for PS exposure after thrombin treatment.