Tetsuya Matoba

Hydrogen peroxide is an endothelium-derived hyperpolarizing factor in mice

The endothelium plays an important role in maintaining vascular homeostasis by synthesizing and releasing several endothelium-derived relaxing factors, such as prostacyclin, nitric oxide (NO), and the previously unidentified endothelium-derived hyperpolarizing factor (EDHF). In this study, we examined our hypothesis that hydrogen peroxide (H(2)O(2)) derived from endothelial NO synthase (eNOS) is an EDHF. EDHF-mediated relaxation and hyperpolarization in response to acetylcholine (ACh) were markedly attenuated in small mesenteric arteries from eNOS knockout (eNOS-KO) mice. In the eNOS-KO mice, vasodilating and hyperpolarizing responses of vascular smooth muscle per se were fairly well preserved, as was the increase in intracellular calcium in endothelial cells in response to ACh. Antihypertensive treatment with hydralazine failed to improve the EDHF-mediated relaxation. Catalase, which dismutates H(2)O(2) to form water and oxygen, inhibited EDHF-mediated relaxation and hyperpolarization, but it did not affect endothelium-independent relaxation following treatment with the K(+) channel opener levcromakalim. Exogenous H(2)O(2) elicited similar relaxation and hyperpolarization in endothelium-stripped arteries. Finally, laser confocal microscopic examination with peroxide-sensitive fluorescence dye demonstrated that the endothelium produced H(2)O(2) upon stimulation by ACh and that the H(2)O(2) production was markedly reduced in eNOS-KO mice. These results indicate that H(2)O(2) is an EDHF in mouse small mesenteric arteries and that eNOS is a major source of the reactive oxygen species.

Cyclophilin A enhances vascular oxidative stress and the development of angiotensin II–induced aortic aneurysms

Inflammation and oxidative stress are pathogenic mediators of many diseases, but molecules that could be therapeutic targets remain elusive. Inflammation and matrix degradation in the vasculature are crucial for abdominal aortic aneurysm (AAA) formation. Cyclophilin A (CypA, encoded by Ppia) is highly expressed in vascular smooth muscle cells (VSMCs), is secreted in response to reactive oxygen species (ROS) and promotes inflammation. Using the angiotensin II (AngII)-induced AAA model in Apoe−/− mice, we show that Apoe−/−Ppia−/− mice are completely protected from AngII–induced AAA formation, in contrast to Apoe−/−Ppia+/+ mice. Apoe−/−Ppia−/− mice show decreased inflammatory cytokine expression, elastic lamina degradation and aortic expansion. These features were not altered by reconstitution of bone marrow cells from Ppia+/+ mice. Mechanistic studies showed that VSMC-derived intracellular and extracellular CypA are required for ROS generation and matrix metalloproteinase-2 activation. These data define a previously undescribed role for CypA in AAA formation and suggest CypA as a new target for treating cardiovascular disease.

Cyclophilin A Is Secreted by a Vesicular Pathway in Vascular Smooth Muscle Cells

Reactive oxygen species (ROS) contribute to the pathogenesis of atherosclerosis in part by promoting vascular smooth muscle cell (VSMC) growth. Previously we demonstrated that cyclophilin A (CyPA) is a secreted oxidative stress-induced factor (SOXF) that promotes inflammation, VSMC growth, and endothelial cell apoptosis. However, the mechanisms that regulate CyPA secretion are unknown. In this study, we hypothesized that ROS-induced CyPA secretion from VSMC requires a highly regulated process of vesicle transport, docking, and fusion at the plasma membrane. Conditioned medium and plasma membrane sheets were prepared by exposing VSMC to 1 &mgr;mol/L LY83583, which generates intracellular superoxide. A vesicular transport mechanism was confirmed by colocalization at the plasma membrane with vesicle-associated membrane protein (VAMP). CyPA transport to the plasma membrane and secretion were significantly increased by LY83583. Reduction of VAMP-2 expression by small interfering RNA inhibited LY83583-induced CyPA secretion. Pretreatment with 3 &mgr;mol/L cytochalasin D, an actin depolymerizing agent, abrogated CyPA secretion. Infection with dominant-negative RhoA and Cdc42 adenovirus inhibited CyPA secretion by 72% and 63%, respectively, whereas dominant-negative Rac1 had a small effect (11%). Pretreatment with the Rho kinase inhibitor Y27632 (3 to 30 &mgr;mol/L) and myosin II inhibitor blebbistatin (1 to 10 &mgr;mol/L) inhibited CyPA secretion in a dose-dependent manner. Simvastatin (3 to 30 &mgr;mol/L) also dose-dependently inhibited LY83583-induced CyPA secretion likely via decreased isoprenylation of small GTPases. Our findings define a novel VSMC vesicular secretory pathway for CyPA that involves actin remodeling and myosin II activation via RhoA-, Cdc42-, and Rho kinase-dependent signaling events.

Extracorporeal cardiac shock wave therapy ameliorates myocardial ischemia in patients with severe coronary artery disease

ObjectivePrognosis of severe coronary artery disease with no indication of percutaneous coronary intervention or coronary artery bypass grafting remains poor. We have recently demonstrated that shock wave therapy effectively induces neovascularization and improves myocardial ischemia in a porcine model in vivo. MethodsWith permission from the Ethical Committee of our Institute, we treated nine patients with end-stage coronary artery disease with no indication of percutaneous coronary intervention or coronary artery bypass grafting (55–82 years old, five men and four women) with our cardiac shock wave therapy (200 shots/spot at 0.09 mJ/mm2 for 20–40 spots, 3 times a week/series). We followed-up the patients at 1, 3, 6, and 12 months after the therapy to examine the amelioration of myocardial ischemia. When needed, shock wave therapy was performed up to three series at 0, and 1, 3 or 6 months. ResultsThe cardiac shock wave therapy improved symptoms (Canadian Cardiovascular Society functional class score, from 2.7±0.2 to 1.8±0.2, P<0.01) and reduced nitroglycerin use (from 5.4±2.5 to 0.3±0.3/week, P<0.05). The treatment also improved myocardial perfusion as assessed by dipyridamole stress thallium scintigraphy (severity score, 25.2±7.2% improvement, P<0.05; extent score, 23.3±9.0% improvement, P=0.10; washout rate, 20±3 to 34±3, P<0.05). Myocardial perfusion was improved only in the ischemic area treated with the therapy. These beneficial effects persisted for 12 months. No procedural complications or adverse effects were noted. ConclusionThese results indicate that our extracorporeal cardiac shock wave therapy is an effective and non-invasive treatment for end-stage coronary artery disease, although further careful evaluation is needed.

Cyclophilin A Mediates Vascular Remodeling by Promoting Inflammation and Vascular Smooth Muscle Cell Proliferation

Background— Oxidative stress, generated by excessive reactive oxygen species, promotes cardiovascular disease. Cyclophilin A (CyPA) is a 20-kDa chaperone protein secreted from vascular smooth muscle cells (VSMCs) in response to reactive oxygen species that stimulates VSMC proliferation and inflammatory cell migration in vitro; however, the role CyPA plays in vascular function in vivo remains unknown. Methods and Results— We tested the hypothesis that CyPA contributes to vascular remodeling by analyzing the response to complete carotid ligation in CyPA knockout mice, wild-type mice, and mice that overexpress CyPA in VSMC (VSMC-Tg). After carotid ligation, CyPA expression in vessels of wild-type mice increased dramatically and was significantly greater in VSMC-Tg mice. Reactive oxygen species–induced secretion of CyPA from mouse VSMCs correlated significantly with intracellular CyPA expression. Intimal and medial hyperplasia correlated significantly with CyPA expression after 2 weeks of carotid ligation, with marked decreases in CyPA knockout mice and increases in VSMC-Tg mice. Inflammatory cell migration into the intima was significantly reduced in CyPA knockout mice and increased in VSMC-Tg mice. Additionally, VSMC proliferation assessed by Ki67+ cells was significantly less in CyPA knockout mice and was increased in VSMC-Tg mice. The importance of CyPA for intimal and medial thickening was shown by strong correlations between CyPA expression and the number of both inflammatory cells and proliferating VSMCs in vivo and in vitro. Conclusions— In response to low flow, CyPA plays a crucial role in VSMC migration and proliferation, as well as inflammatory cell accumulation, thereby regulating flow-mediated vascular remodeling and intima formation.

Formulation of nanoparticle-eluting stents by a cationic electrodeposition coating technology: efficient nano-drug delivery via bioabsorbable polymeric nanoparticle-eluting stents in porcine coronary arteries.

OBJECTIVES The objective of this study was to formulate a nanoparticle (NP)-eluting drug delivery stent system by a cationic electrodeposition coating technology. BACKGROUND Nanoparticle-mediated drug delivery systems (DDS) are poised to transform the development of innovative therapeutic devices. Therefore, we hypothesized that a bioabsorbable polymeric NP-eluting stent provides an efficient DDS that shows better and more prolonged delivery compared with dip-coating stent. METHODS We prepared cationic NP encapsulated with a fluorescence marker (FITC) by emulsion solvent diffusion method, succeeded to formulate an NP-eluting stent with a novel cation electrodeposition coating technology, and compared the in vitro and in vivo characteristics of the FITC-loaded NP-eluting stent with dip-coated FITC-eluting stent and bare metal stent. RESULTS The NP was taken up stably and efficiently by cultured vascular smooth muscle cells in vitro. In a porcine coronary artery model in vivo, substantial FITC fluorescence was observed in neointimal and medial layers of the stented segments that had received the FITC-NP-eluting stent until 4 weeks. In contrast, no substantial FITC fluorescence was observed in the segments from the polymer-based FITC-eluting stent or from bare metal stent. The magnitudes of stent-induced injury, inflammation, endothelial recovery, and neointima formation were comparable between bare metal stent and NP-eluting stent groups. CONCLUSIONS Therefore, this NP-eluting stent is an efficient NP-mediated DDS that holds as an innovative platform for the delivery of less invasive nano-devices targeting cardiovascular disease.

Inhibition of Renin-Angiotensin System Ameliorates Endothelial Dysfunction Associated With Aging in Rats

Objective—Endothelial vasodilator functions are progressively impaired with aging, which may account in part for the increased incidence of cardiovascular events in elderly people. We examined what treatment could ameliorate the endothelial dysfunction associated with aging in rats. Methods and Results—Aged (12-month-old) Wistar-Kyoto rats were treated with vehicle, temocapril, CS-866 (an angiotensin II type 1 receptor antagonist), cerivastatin, or hydralazine for 2 weeks. Endothelium-dependent relaxations (EDRs) of aortas from aged rats were markedly impaired compared with EDRs of aortas from young (3-month-old) rats. Indomethacin, NS-398 (a cyclooxygenase [COX]-2 inhibitor), and SQ-29548 (a thromboxane A2/prostaglandin H2 receptor antagonist) acutely restored EDRs in aged rats, suggesting an involvement of COX-2–derived vasoconstricting eicosanoids. Tiron, a superoxide scavenger, also partially improved EDRs, suggesting an involvement of superoxide. EDRs were significantly ameliorated in aged rats after long-term treatment with temocapril or CS-866 but not after treatment with cerivastatin or hydralazine. Indomethacin induced no further improvement of EDRs after treatment with temocapril or CS-866. COX-2 protein expression and superoxide production were increased in the aortas of aged rats and were also attenuated by treatment with temocapril or CS-866. Conclusions—These results demonstrate that long-term inhibition of the renin-angiotensin system ameliorates endothelial dysfunction associated with aging through the inhibition of the synthesis of COX-2–derived vasoconstricting factors and superoxide anions.

Usefulness of fasudil, a Rho-kinase inhibitor, to treat intractable severe coronary spasm after coronary artery bypass surgery.

We have recently demonstrated that fasudil, a Rho-kinase inhibitor, is effective in suppressing coronary artery spasm in patients with vasospastic angina. Thus, blockade of Rho-kinase may provide a novel therapeutic strategy to treat ischemic coronary syndrome caused by the spasm. Severe coronary artery spasm still remains a life-threatening serious complication of coronary artery bypass grafting (CABG). In this study, we examined the inhibitory effect of fasudil in patients with intractable severe coronary spasm after CABG. Three patients who underwent CABG showed severe myocardial ischemia resistant to intensive therapy with intravenous conventional vasodilators, including isosorbide dinitrate (ISDN), diltiazem, and nicorandil. Coronary angiography revealed severe coronary spasm in native coronary arteries and/or bypass arterial grafts in all patients. Since intracoronary and/or intragraft administration of ISDN was ineffective to resolve the spasm, we then administered fasudil (1.5 mg/min for 15 minutes) into the spastic arteries. Fasudil successfully resolved the spasm and improved myocardial ischemia in all patients without any systemic adverse effects. In conclusion, the treatment with fasudil may be useful to treat intractable and otherwise fatal coronary spasm resistant to intensive conventional vasodilator therapy after CABG.

Influence of diabetes mellitus, hypercholesterolemia, and their combination on EDHF-mediated responses in mice

The endothelium synthesizes and releases several vasodilator substances, including vasodilator prostaglandins, NO, and EDHF. NO-mediated relaxations are reduced by various risk factors, such as diabetes mellitus and hypercholesterolemia. However, it remains to be elucidated whether EDHF-mediated relaxations also are reduced by those factors and their combination. In this study, we addressed this point in mice. We used small mesenteric arteries from control, diabetic (streptozotocin-induced), apolipoprotein-E-deficient (ApoE−/−), and diabetic ApoE−/− mice. In control mice, endothelium-dependent relaxations to acetylcholine were largely mediated by EDHF. This EDHF-mediated component was slightly reduced in diabetic mice, preserved in ApoE−/− mice, and markedly reduced in diabetic ApoE−/− mice with an increase in NO-mediated component and a negative contribution of indomethacin-sensitive endothelium-derived contracting factor (EDCF). Endothelium-independent relaxations to sodium nitroprusside or NS1619, a direct opener of calcium-activated K channels, were attenuated in ApoE−/− and diabetic ApoE−/− mice. Endothelium-dependent hyperpolarizations were significantly reduced in diabetic mice, preserved in ApoE−/− mice, and again markedly reduced in diabetic ApoE−/− mice. These results indicate that hypercholesterolemia alone minimally affects the EDHF-mediated relaxations, and diabetes mellitus significantly attenuated the responses, whereas their combination markedly attenuates the responses with a compensatory involvement of NO and a negative contribution of EDCF.

Nanoparticle-Mediated Delivery of Pitavastatin Inhibits Atherosclerotic Plaque Destabilization/Rupture in Mice by Regulating the Recruitment of Inflammatory Monocytes

Background— Preventing atherosclerotic plaque destabilization and rupture is the most reasonable therapeutic strategy for acute myocardial infarction. Therefore, we tested the hypotheses that (1) inflammatory monocytes play a causative role in plaque destabilization and rupture and (2) the nanoparticle-mediated delivery of pitavastatin into circulating inflammatory monocytes inhibits plaque destabilization and rupture. Methods and Results— We used a model of plaque destabilization and rupture in the brachiocephalic arteries of apolipoprotein E–deficient (ApoE−/−) mice fed a high-fat diet and infused with angiotensin II. The adoptive transfer of CCR2+/+Ly-6Chigh inflammatory macrophages, but not CCR2−/− leukocytes, accelerated plaque destabilization associated with increased serum monocyte chemoattractant protein-1 (MCP-1), monocyte-colony stimulating factor, and matrix metalloproteinase-9. We prepared poly(lactic-co-glycolic) acid nanoparticles that were incorporated by Ly-6G−CD11b+ monocytes and delivered into atherosclerotic plaques after intravenous administration. Intravenous treatment with pitavastatin-incorporated nanoparticles, but not with control nanoparticles or pitavastatin alone, inhibited plaque destabilization and rupture associated with decreased monocyte infiltration and gelatinase activity in the plaque. Pitavastatin-incorporated nanoparticles inhibited MCP-1–induced monocyte chemotaxis and the secretion of MCP-1 and matrix metalloproteinase-9 from cultured macrophages. Furthermore, the nanoparticle-mediated anti–MCP-1 gene therapy reduced the incidence of plaque destabilization and rupture. Conclusions— The recruitment of inflammatory monocytes is critical in the pathogenesis of plaque destabilization and rupture, and nanoparticle-mediated pitavastatin delivery is a promising therapeutic strategy to inhibit plaque destabilization and rupture by regulating MCP-1/CCR2–dependent monocyte recruitment in this model.