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Yuko Furusho,
Masaaki Miyata,
Takami Matsuyama,
Taku Nagai,
Hua Li, Yuichi Akasaki,
Narisato Hamada,
Takahiro Miyauchi,
Yoshiyuki Ikeda,
Takahiro Shirasawa,
Kanako Ide,
Chuwa Tei
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ABSTRACT: Folate receptor β (FRβ) is induced during macrophage activation. A recombinant immunotoxin consisting of the truncated Pseudomonas exotoxin A (PE38) conjugated to an anti-FRβ antibody (anti-FRβ-PE38) has been reported to kill activated macrophages in inflammatory diseases. To elucidate the effect of an immunotoxin targeting FRβ on atherosclerosis, we determined the presence of FRβ-expressing macrophages in atherosclerotic lesions and administered the FRβ immunotoxin in apolipoprotein E-deficient mice.
The FRβ-expressing macrophages were observed in atherosclerotic lesions of apolipoprotein E-deficient mice. At 15 or 35 weeks of age, the apolipoprotein E-deficient mice were divided into 3 groups and were intravenously administered 0.1 mg/kg of anti-FRβ-PE38 (immunotoxin group), 0.1 mg/kg of PE38 (toxin group), or 0.1 mL of saline (control group) every 3 days, for a total of 5 times for each age group. The mice were analyzed at 21 or 41 weeks of age. Treatment with the immunotoxin resulted in 31% and 22% reductions in atherosclerotic lesions of the 21- and 41-week-old mice, respectively (P<0.05). Administration of immunotoxin reduced the numbers of FRβ- and tumor necrosis factor-α-expressing macrophages, reduced cell proliferation, and increased the number of apoptotic cells (P<0.05). Real-time polymerase chain reaction demonstrated that the expression of FRβ and tumor necrosis factor-α mRNA was significantly decreased in the immunotoxin group (P<0.05).
These results suggest that FRβ-expressing macrophages exist in the atherosclerotic lesions of apolipoprotein E-deficient mice and that FRβ immunotoxin administration reduces the progression of atherosclerotic lesions in younger and older individuals. The recombinant FRβ immunotoxin targeting activated macrophages could provide a novel therapeutic tool for atherosclerosis. (J Am Heart Assoc. 2012;1:e003079 doi: 10.1161/JAHA.112.003079.).
Journal of the American Heart Association. 08/2012; 1(4):e003079.
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ABSTRACT: Thermal therapy, namely Waon therapy, has previously been reported to regulate nitric oxide (NO) and endothelial NO synthase (eNOS) and augment ischemia-induced angiogenesis in mice and improve limb ischemia in patients with peripheral artery disease. The aim of this study was to clarify the precise mechanism by which Waon therapy augments angiogenesis in mice with hindlimb ischemia.
Unilateral hindlimb ischemia was induced in apolipoprotein E-deficient mice and Waon therapy was performed for 5 weeks. Heat shock protein 90 (Hsp90), phosphorylated-Akt, and phosphorylated-eNOS were detected in arterial endothelial cells of ischemic hindlimbs and all were upregulated by Waon therapy compared to controls. Waon therapy also increased serum concentrations of nitrite and nitrate. Capillary density and the ischemic limb/normal side blood perfusion ratio monitored by laser Doppler perfusion imaging in the Waon therapy group were significantly increased beyond those in the control group. The effect of Waon therapy on angiogenesis through the activation of the Hsp90/Akt/eNOS pathway was attenuated by the administration of a Hsp90 inhibitor.
It is suggested that Waon therapy upregulates Hsp90, which contributes to the activation of the Akt/eNOS/NO pathway, and induces angiogenesis in mice with hindlimb ischemia.
Circulation Journal 04/2012; 76(7):1712-21. · 3.77 Impact Factor
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Narisato Hamada,
Masaaki Miyata,
Hideyuki Eto,
Yoshiyuki Ikeda,
Takahiro Shirasawa, Yuichi Akasaki,
Takahiro Miyauchi,
Yuko Furusho,
Aya Nagaki,
Bruce Jefferson Aronow,
Chuwa Tei
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ABSTRACT: Whether clusterin/apolipoprotein J is antiatherogenic or proatherogenic is controversial. We reported that clusterin was markedly induced in media and neointima after vascular injury and that reduced clusterin expression reduced the proliferation of vascular smooth muscle cells (VSMCs), which induced G1 arrest via p53 and p21. The purpose of this study was to investigate the physiological function of clusterin in atherosclerosis using double-knockout mice (D-KO) of apolipoprotein E-deficient mice (apoE-KO) and clusterin-deficient mice (CLU-KO).
Atherosclerotic lesions in the aortic root were quantitated at 20 weeks of age. Atherosclerotic lesions of D-KO were significantly smaller than those of apoE-KO (D-KO: 0.176±0.078 mm(2) vs. apoE-KO: 0.365±0.164 mm(2), p< 0.001). To identify underlying atherosclerotic mechanisms that were blocked by loss of clusterin, we performed immunohistochemical analysis of Egr-1. Egr-1 immunoreactivity in the nuclei of VSMCs in atherosclerotic lesions of apoE-KO was upregulated, whereas it was not in D-KO lesions. Western blotting demonstrated that the expression levels of Egr-1 and TNF-α in the D-KO were significantly lower than those in the apoE-KO. When VSMCs and macrophages were obtained from D-KO and apoE-KO, Western blotting showed that the expression levels of Egr-1 and TNF-α in VSMCs and macrophages of D-KO were significantly lower than those of apoE-KO.
Loss of clusterin strongly suppressed apoE-KO-induced atherosclerotic lesions at a step prior to the expression of Egr-1 and TNF-α, suggesting that clusterin is a candidate for an antiatherogenic target.
Journal of atherosclerosis and thrombosis 11/2010; 18(3):209-16. · 2.69 Impact Factor
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ABSTRACT: We previously reported that Waon therapy upregulates endothelial nitric oxide synthase protein, and augments ischemia-induced angiogenesis in mice with hindlimb ischemia, and it improves limb ischemia in patients with peripheral arterial disease (PAD). The aim of this study was to investigate the underlying mechanism of Waon therapy for the treatment of patients with PAD, and to determine whether Waon therapy can mobilize blood-derived progenitor cells.
21 consecutive PAD patients received standard medications, and were randomly divided into control (n=10) and Waon therapy groups (n=11). The Waon therapy group received Waon therapy daily for 6 weeks. The control group continued conventional therapy for 6 weeks. Leg pain was scored using a visual analogue scale. The ankle-brachial pressure index (ABPI) and the 6-min walking distance were measured at baseline and 6 weeks after therapy. Frequency of circulating CD34+ progenitor cell numbers was measured by quantitative real-time polymerase chain reaction, and the serum nitrate and nitrite levels were also measured at baseline and 6 weeks after therapy.
The leg pain score, ABPI and the 6-min walking distance improved significantly after 6 weeks in the Waon therapy group, but not in the control group. Frequency of circulating CD34+ cells increased after 6 weeks of Waon therapy [2.0 ± 1.2 (×10(-4)) at baseline to 3.9 ± 1.9 (×10(-4)), p=0.015], while it remained unchanged in the control group [1.8 ± 1.8 (×10(-4)) at baseline to 1.2 ± 0.9 (×10(-4))]. Serum nitrate and nitrite levels increased significantly after Waon therapy (29.6 ± 17.6 to 36.0 ± 17.7 μmol/ml, p<0.05), but not in the control group (34.4 ± 9.4 to 38.3 ± 8.8 μmol/ml).
Waon therapy mobilized circulating endothelial progenitor cells and improved limb ischemia in patients with PAD. Waon therapy is a highly promising therapy for patients with PAD.
Journal of Cardiology 11/2010; 56(3):361-6. · 1.28 Impact Factor
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ABSTRACT: The purpose is to elucidate the mechanism by which a newly developed tacrolimus-eluting stent (TES) prevents neointimal hyperplasia after stenting.
The three major coronary arteries in juvenile swine were randomized to implantation of either a TES or bare metal stent (BMS). Twelve weeks after stenting, the TES showed 29% less neointimal area than the BMS. Immunohistochemical staining showed that the expression of calcineurin was up-regulated in the neointima and media after stenting, and the TES inhibited this up-regulation. Western blotting demonstrated that the expression of calcineurin, nuclear factor of activated T cell (NFAT), and interleukin-2 (IL-2) was lower with the TES than with the BMS. To confirm the effect of tacrolimus on vascular smooth muscle cells (VSMCs) and its mechanism, cultured rat VSMCs were incubated with 12.5 microM of tacrolimus (tacrolimus group) or without tacrolimus (control group). The cell number of the tacrolimus group was significantly lower than that of the control group at 48 h of incubation. Western blotting demonstrated that tacrolimus decreased the expression of calcineurin, NFATc4, and IL-2 of cultured VSMCs. We confirmed that calcineurin small-interfering RNA (siRNA) decreased cell proliferation and the expression of NFATc4 and IL-2 in cultured VSMCs compared with negative control-siRNA.
The newly developed TES inhibited neointimal hyperplasia after stenting via the calcineurin/NFAT/IL-2 signaling pathway, which is one of several mechanisms through which TES inhibits restenosis. Calcineurin may be an important molecular target to prevent restenosis after stenting.
Atherosclerosis 08/2009; 208(1):97-103. · 3.79 Impact Factor
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Takahiro Shirasawa,
Masaaki Miyata,
Hideyuki Eto,
Narisato Hamada, Yuichi Akasaki,
Takahiro Miyauchi,
Yuko Furusho,
Koji Orihara,
Shuichi Hamasaki,
Bruce J Aronow,
Jonathan D Smith,
Chuwa Tei
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ABSTRACT: Increased clusterin mRNA and protein levels have been detected in various tissues undergoing stress, and we previously reported that clusterin is markedly induced in media and neointima following vascular injury. The present study therefore investigated the impact of clusterin on neointimal hyperplasia following vascular injury.
As compared with wild-type mice, clusterin knockout mice (clusterin-KO) demonstrated a significant decrease of the intima/media ratio 4 weeks after cuff placement. Immunohistochemical analysis of injured femoral arteries in clusterin-KO demonstrated the accumulation of p53 in nuclei of neointimal vascular smooth muscle cells (VSMCs). Moreover, VSMCs from either clusterin-KO or rat VSMCs treated with clusterin-short-interfering (si) RNA subjected to static stretch exhibited significantly increased p53 and p21, and increased G1 cell cycle arrest as indicated by flow cytometry compared with VSMCs from wild-type mice.
Reduced clusterin expression reduced the proliferation of VSMCs and induced G1 arrest via p53 and p21. Clusterin therefore represents a promising molecular target to limit restenosis after coronary intervention.
Journal of atherosclerosis and thrombosis 01/2009; 16(6):772-81. · 2.69 Impact Factor
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ABSTRACT: Angiotensin II type 1 receptor may contribute to atherogenesis by facilitating the proliferative and inflammatory response to hypercholesterolemia. In the present study, we investigated the long-term effect of angiotensin II type 1a receptor (AT1a) deficiency on hypercholesterolemia-induced atherosclerosis by the use of AT1a-knockout (AT1a-KO) mice and apolipoprotein E-knockout (apoE-KO) mice. AT1a-KO were crossed with apoE-KO, generating double-knockout (D-KO) mice. Mice were fed a standard diet and analyzed at 25- or 60-weeks-old. The quantification of atherosclerotic volume in the aortic root revealed that the atherosclerotic lesions of D-KO mice were significantly smaller than those of apoE-KO mice at 25-week-old (0.81+/-0.16 mm2 vs. 1.05+/-0.21 mm2, p<0.001) and at 60-week-old (0.89+/-0.11 mm2 vs. 2.44+/-0.28 mm2, p<0.001). Surprisingly, there was no significant difference in atherosclerotic lesion size of D-KO mice at 25- and 60-week-old, suggesting that AT1a deficiency completely protected against the age-related progression of atherosclerosis. The amounts of collagen and elastin, the expression of p22phox, serum amyloid P (SAP), matrix metalloproteinase (MMP)-2, and MMP-9, and the number of apoptotic cells of D-KO mice were lower than those of apoE-KO mice. Furthermore, we confirmed that the expression of procollagen alpha1(I), procollagen alpha1(III), tropoelastin, p22phox, SAP, MMP-2, and MMP-9 decreased in cultured vascular smooth muscle cells from D-KO mice compared with those of apoE-KO mice. In conclusion, AT1a deficiency reduces atherosclerotic lesion size of apoE-KO mice and protects against the age-related progression of atherosclerosis. Reduction of oxidative stress, apoptosis, and MMP expression in atherosclerotic lesions by AT1a deficiency may contribute to plaque size.
Hypertension Research 09/2008; 31(8):1631-42. · 2.58 Impact Factor
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ABSTRACT: Nitric oxide (NO), constitutively produced by endothelial NO synthase (eNOS), plays roles in angiogenesis. Having reported that thermal therapy up-regulated the expression of arterial eNOS in hamsters, we investigated whether this therapy increased angiogenesis in mice with hindlimb ischemia.
Unilateral hindlimb ischemia was induced in apolipoprotein E-deficient mice, which were divided into control and thermal therapy groups. The latter mice were placed in a far-infrared dry sauna at 41 degrees C for 15 min and then at 34 degrees C for 20 min once daily for 5 weeks. Laser Doppler perfusion imaging demonstrated that the ischemic limb/normal side blood perfusion ratio in the thermal therapy group was significantly increased beyond that in controls (0.79+/-0.04 vs 0.54+/-0.08, p<0.001). Significantly greater capillary density was seen in thermal therapy group (757+/-123 /mm2 vs 416+/-20 /mm2, p<0.01). Western blotting showed thermal therapy markedly increased hindlimb eNOS expression. To study possible involvement of eNOS in thermally induced angiogenesis, thermal therapy was given to mice with hindlimb ischemia with or without N(G)-nitro-L-arginine methyl ester (L-NAME) administration for 5 weeks. L-NAME treatment eliminated angiogenesis induced using thermal therapy. Thermal therapy did not increase angiogenesis in eNOS-deficient mice.
Angiogenesis was induced via eNOS using thermal therapy in mice with hindlimb ischemia.
Circulation Journal 04/2006; 70(4):463-70. · 3.77 Impact Factor
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Hiroshi Fujiwara,
Naomichi Arima, Yuichi Akasaki,
Hideo Ohtsubo,
Atsuo Ozaki,
Toshimasa Kukita,
Kakushi Matsushita,
Kosei Arimura,
Yukio Suruga,
Shinichi Wakamatsu,
Tadashi Matsumoto,
Shiroh Hidaka,
Yoshito Eizuru,
Chuwa Tei
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ABSTRACT: In the present report, we describe a case of adult T cell leukemia/lymphoma (ATLL), a 58-year-old woman, successfully treated with interferon (IFN)-alpha following autologous peripheral blood stem cell transplantation (auto-PBSCT). The patient remains in remission with full performance status for more than 12 months. Auto-PBSCT reduced the abdominal lymphoma mass and IFN-alpha eliminated residual tumor cells, possibly through the induction of specific T-cell subsets expressing CD3, CD8 on their surfaces and either IFN-gamma or tumor necrosis factor (TNF)-alpha in cytoplasm. We have treated a total of 4 ATLL patients with auto-PBSCT, including the case presented herein. All other patients treated with auto-PBSCT were not followed by adjuvant chemotherapy or cytokine therapy and relapsed within 3 months after auto-PBSCT. This evidence suggests that the therapeutic success of the present case was attributable to the administration of IFN-alpha immunotherapy following auto-PBSCT.
Acta Haematologica 02/2002; 107(4):213-9. · 1.35 Impact Factor
