Biomedical subjects

Yuji Kashiwakura

Publications and source records attributed to Yuji Kashiwakura.

10 recordsLinked to original sources

PubMed2006

Gene transfer of a synthetic pacemaker channel into the heart: a novel strategy for biological pacing.

BACKGROUND: One key element of natural pacemakers is the pacemaker current encoded by the hyperpolarization-activated nucleotide-gated channel (HCN) gene family. Although HCN gene transfer has been used to engineer biological pacemakers, this strategy may be confounded by unpredictable consequences of heteromultimerization with endogenous HCN family members and limited flexibility with regard to frequency tuning of the engineered pacemaker. METHODS AND RESULTS: To circumvent these limitations, we converted a depolarization-activated potassium-selective channel, Kv1.4, into a hyperpolarization-activated nonselective channel by site-directed mutagenesis (R447N, L448A, and R453I in S4 and G528S in the pore). Gene transfer into ventricular myocardium demonstrated the ability of this construct to induce pacemaker activity with spontaneous action potential oscillations in adult ventricular myocytes and idioventricular rhythms by in vivo electrocardiography. CONCLUSIONS: Given the sparse expression of Kv1 family channels in the human ventricle, gene transfer of a synthetic pacemaker channel based on the Kv1 family has novel therapeutic potential as a biological alternative to electronic pacemakers.

Animals↗

PubMed2006

Efficient expression of a transgene in platelets using simian immunodeficiency virus-based vector harboring glycoprotein Ibalpha promoter: in vivo model for platelet-targeting gene therapy.

Platelets release several mediators that modify vascular integrity and hemostasis. In the present study, we developed a technique for efficient transgene expression in platelets in vivo and examined whether this targeted-gene-product delivery system using a platelet release reaction could be exploited for clinical applications. Analysis of luciferase reporter gene constructs driven by platelet-specific promoters (the GPIIb, GPIbalpha, and GPVI) revealed that the GPIbalpha promoter was the most potent in the megakaryoblastic cell line UT-7/TPO and human CD34+-derived megakaryocytes. Transduction of UT-7/TPO; CD34+-derived megakaryocytes; and c-Kit+, ScaI+, and Lineage- (KSL) murine hematopoietic stem cells with a simian immunodeficiency virus (SIV)-based lentiviral vector carrying eGFP resulted in efficient, dose-dependent expression of eGFP, and the GPIbalpha promoter seemed to bestow megakaryocytic-specific expression. Transplantation of KSL cells transduced with SIV vector containing eGFP into mice showed that there was preferable expression of eGFP in platelets driven by the GPIbalpha promoter [7-11% for the cytomeglovirus (CMV) promoter, 16-27% for the GPIbalpha promoter]. Furthermore, transplantation of ex vivo-transduced KSL cells by SIV vector carrying human factorVIII (hFVIII) driven by the GPIbalpha promoter induced the production of detectable transcripts of the hFVIII gene and the hFVIII antigen in bone marrow and spleen for at least 90 days and partially corrected the hemophilia A phenotype. Platelet-targeting gene therapy using SIV vectors appears to be promising for gene therapy approaches toward not only inherited platelet diseases but also other hemorrhagic disorders such as hemophilia A.

Animals↗

PubMed2005

Phenotype correction of hemophilia A mice with adeno-associated virus vectors carrying the B domain-deleted canine factor VIII gene.

Adeno-associated virus (AAV) vectors carrying the B domain-deleted canine FVIII (BDD cFVIII) gene utilizing the beta-actin minimum promoter (167b) pseudotyped with serotype 1 (AAV1-beta-actin-cFVIII) and serotype 8 (AAV8-beta-actin-cFVIII) were developed to express cFVIII in hemophilia A mice. FVIII clotting activities measured by the APTT method increased in hemophilia A mice with intramuscular injection of AAV1-beta-actin-cFVIII in a dose-dependent manner. Therapeutic FVIII levels (2.9+/-1.0%) in hemophilia A mice with the AAV1-beta-actin-cFVIII dose of 1 x 10(12) gc/body were achieved, suggesting partial correction of the phenotype with AAV1-beta-actin-cFVIII vectors. FVIII clotting activity levels in hemophilia A mice with intravenous injection of AAV8-beta-actin-cFVIII also were increased dose-dependently, achieving therapeutic FVIII levels (5-90%) in hemophilia A mice with the AAV8-beta-actin-cFVIII doses of 1-3 x 10(11) gc/body and supernormal FVIII levels (180-670%) in hemophilia A mice with the AAV8-beta-actin-cFVIII dose of 1 x 10(12) gc/body. Transduction of the liver with AAV8-beta-actin-cFVIII is superior to transduction of skeletal muscles with AAV1cFVIII regarding the FVIII production and antibody formation. These data suggested that both AAV1 and AAV8 vectors carrying the FVIII gene utilizing a minimum promoter have a potential for hemophilia A gene therapy.

Adenoviridae↗

PubMed2005

Severe secondary deficiency of von Willebrand factor-cleaving protease (ADAMTS13) in patients with sepsis-induced disseminated intravascular coagulation: its correlation with development of renal failure.

Deficiency of ADAMTS13 is found in patients with thrombotic thrombocytopenic purpura (TTP), and the genetic defects in the ADAMTS13 gene or the autoantibody against ADAMTS13 is thought to be responsible for the development of TTP. The clinical correlation and mechanisms of secondary ADAMTS13 deficiency in other disease states were investigated. In addition to TTP, ADAMTS13 levels were severely decreased in patients with sepsis-induced disseminated intravascular coagulation (DIC). The incidence of acute renal failure and serum creatinine levels in patients with ADAMTS13 activity levels lower than 20% (incidence, 41.2%; creatinine, 160 +/- 150 microM [1.81 +/- 1.70 mg/dL]) (P < .05) were significantly higher than they were in patients with ADAMTS13 activity levels higher than 20% (incidence, 15.4%; creatinine, 84 +/- 67 microM [0.95 +/- 0.76 mg/dL]) (P < .01). Additionally, unusually large von Willebrand factor multimers were detected in 26 (51.0%) of 51 patients with ADAMTS13 activity levels lower than 20%. Lower molecular weight forms of ADAMTS13 were found in the plasma of patients with sepsis-induced DIC, suggesting that the deficiency of ADAMTS13 was partially caused by its cleavage by proteases in addition to decreased synthesis in the liver. These data suggested that severe secondary ADAMTS13 deficiency can be associated with sepsis-induced DIC and may contribute to the development of renal failure.

ADAM Proteins↗

PubMed2005

Adeno-associated virus 2-mediated intratumoral prostate cancer gene therapy: long-term maspin expression efficiently suppresses tumor growth.

Maspin is a member of the serine protease inhibitors and the maspin gene, a tumor suppressor gene, is down-regulated in a large fraction of prostate cancers. We evaluated the use of adeno-associated virus (AAV, serotype 2) vector encoding maspin as a means for in vivo gene therapy for human prostate cancer. TUNEL assay of subcutaneously formed LNCaP or DU145 tumors in nude mice showed that intratumoral AAV-mediated maspin expression significantly upregulated the number of apoptotic cells compared with AAV-LacZ treatment. Immunofluorescence double staining for maspin protein and apoptosis in LNCaP tumors showed that the percentage of apoptotic cells in AAV-maspin-mediated maspin-expressing cells was significantly high compared with that in AAV-GFP-mediated GFP-expressing cells. Moreover, significantly fewer CD31-positive microvessels were observed in AAV-maspin-treated tumors compared with the control tumors. These therapeutic responses were highly correlated to persistent maspin expression in tumors, confirmed by Western blot analysis until at least day 56 after treatment. Finally, intratumoral delivery of AAV-maspin significantly suppressed growth of LNCaP and DU145 tumors and improved survival of mice. We conclude that AAV-mediated prolonged maspin expression efficiently suppresses human prostate tumor growth in vivo by apoptosis induction and inhibition of angiogenesis.

Animals↗

PubMed2005

Hepatocyte growth factor receptor is a coreceptor for adeno-associated virus type 2 infection.

After the first attachment of virus to the cell surface through a primary receptor, efficient entry of virus requires the presence of a coreceptor. For adeno-associated virus type 2 (AAV2) infection, heparan sulfate proteoglycan is supposed as the primary receptor, and alphavbeta5 integrin and FGFR1 are reported to act as coreceptors. In this study, we were able to demonstrate that hepatocyte growth factor receptor, c-Met, is also a coreceptor for AAV2 infection. AAV2-mediated transgene analyses revealed that c-Met expression significantly up-regulated transgene expression without increasing AAV2 cell binding. Moreover, a viral overlay assay elucidated the physical interaction between AAV2 and the beta subunit of c-Met. These data suggest that c-Met plays the role of coreceptor for AAV2 infection by facilitating AAV2 internalization into the cytoplasm.

3T3 Cells↗

PubMed2004

Dynamin-2 regulates oxidized low-density lipoprotein-induced apoptosis of vascular smooth muscle cell.

BACKGROUND: On exposure to oxidized low-density lipoprotein (oxLDL), vascular cells generally undergo apoptosis, which is one of the major pathogenic factors of atherosclerosis. In this study, we examined the role of dynamin (a crucial GTPase protein in endocytosis) in oxLDL-induced apoptosis of vascular smooth muscle cells (VSMC). METHODS AND RESULTS: After oxLDL stimulation, dynamin-2 colocalized with LOX-1 around the cell surface, as well as oxLDL in the cytoplasm, suggesting that dynamin-2 was involved in scavenger receptor-mediated oxLDL endocytosis. Downregulation of dynamin-2 induced by dynamin-2 dominant negative plasmid (K44A) resulted in a decrease of oxLDL uptake and thereby in a reduction of apoptosis. These data demonstrated that dynamin-2 was involved in oxLDL-induced apoptosis via the oxLDL endocytotic pathway. On the other hand, dynamin-2 wild-type plasmid transfection promoted oxLDL-induced apoptosis without increasing oxLDL uptake. Interestingly, the p53 inhibitor pifithrin-alpha (PFT) significantly reduced apoptosis promoted by wild-type dynamin-2 (78% reduction compared with the PFT[-] condition). These results indicated that dynamin-2 enhanced oxLDL-induced apoptosis of VSMC by participating in the p53 pathway, probably as a signal transducer. Moreover, we demonstrated that, in advanced plaques of apolipoprotein E-/- mice, dynamin-2 expression was often enhanced in apoptotic VSMC, suggesting that dynamin-2 might participate in apoptosis of VSMC even in vivo. CONCLUSIONS: Our data demonstrated that dynamin-2 at least partially regulated oxLDL-induced apoptosis of VSMC by participating in 2 independent pathways: the oxLDL endocytotic pathway and the p53 pathway. These findings suggest that dynamin-2 may serve as a new research or therapeutic target in vascular disease.

Animals↗

PubMed2004

Thrombophilic dysfibrinogen Tokyo V with the amino acid substitution of gammaAla327Thr: formation of fragile but fibrinolysis-resistant fibrin clots and its relevance to arterial thromboembolism.

Thrombophilic dysfibrinogen Tokyo V was identified in a 43-year-old man with recurrent thromboembolism. Based on analyses of the patient fibrinogen genes, the amino acid sequence of the aberrant fibrinogen peptide, and deglycosylation experiments, fibrinogen Tokyo V was shown to have an amino acid substitution of gamma Ala327Thr and possibly extra glycosylation at gamma Asn325 because the mutation confers the N-linked glycosylation consensus sequence Asn-X-Thr. The mutation resulted in impaired function and hypofibrinogenemia (hypodysfibrinogen). Polymerization of fibrin monomers derived from patient fibrinogen was severely impaired with a partial correction in the presence of calcium, resulting in very low clottability. Additionally, a large amount of soluble cross-linked fibrin was formed upon thrombin treatment in the presence of factor XIII and calcium. However, Tokyo V-derived fibrin was resistant to degradation by tissue plasminogen activator (tPA)-catalyzed plasmin digestion. The structure of Tokyo V fibrin appeared severely perturbed, since there are large pores inside the tangled fibrin networks and fiber ends at the boundaries. Taken together, these data suggest that Tokyo V fibrin clots are fragile, so that fibrinolysis-resistant insoluble fibrin and soluble fibrin polymers may be released to the circulation, partly accounting for the recurrent embolic episodes in the patient.

Adult↗

PubMed2003

Isolation of bone marrow stromal cell-derived smooth muscle cells by a human SM22alpha promoter: in vitro differentiation of putative smooth muscle progenitor cells of bone marrow.

BACKGROUND: Bone marrow stromal cells (BMSCs) have many characteristics of mesenchymal stem cells that can differentiate into smooth muscle cells (SMCs). However, there have been few studies closely following the cell development of smooth muscle lineage among BMSCs. METHODS AND RESULTS: To investigate the possible existence of a cell population committed to the SMC lineage among bone marrow adhesion cells, we tried to detect and follow the in vitro differentiation of such a cell type by using a promoter-sorting method with a human SM22alpha promoter (-480 bp)/green fluorescent protein (GFP) construct. The construct was transfected to adhesion cells that appeared 5 days after the seeding of mononuclear cells from bone marrow. GFP was first detectable 5 days after the transfection in a cell population [Ad(G) cells], which expressed PDGF-beta but neither mature (calponin) nor immature (SMemb) SMC-specific proteins at that time. However, the cells were eventually grown into individual clones that expressed SMC-specific proteins (alpha-smooth muscle actin, calponin, and SM-1), suggesting that Ad(G) cells have partly at least progenitor properties. Because early studies have reported that PDGF-beta signaling plays pivotal roles in the differentiation of mesenchymal smooth muscle progenitor cells, Ad(G) cells might be putative mesenchymal smooth muscle progenitors expressing PDGF-beta. CONCLUSIONS: We demonstrated the presence of a cell population fated to become SMCs and followed their differentiation into SMCs among BMSCs.

Animals↗

PubMed2002

Platelets activated by collagen through immunoreceptor tyrosine-based activation motif play pivotal role in initiation and generation of neointimal hyperplasia after vascular injury.

BACKGROUND: Platelet adhesion on components of the extracellular matrix and platelet activation by those components are crucial for the arrest of posttraumatic bleeding, but they can also harm tissue by occluding diseased vessels. Recent studies have shown that the activation of platelets by collagen is mediated through the same pathway used by immune receptors, with an immunoreceptor tyrosine-based activation motif on the Fc receptor gamma chain (FcRgamma) playing a pivotal role. METHODS AND RESULTS: We examined the role of collagen-stimulated platelets in the development of injury-induced neointimal formation by using mice deficient in FcRgamma. The left femoral arteries of 8- to 12-week-old FcRgamma-deficient mice (n=16) and C57BL/6 (wild-type) mice (n=16) were injured by a straight spring wire (0.35-mm diameter). Segments of the injured and uninjured femoral arteries were excised at 7 days and 28 days after the vascular injury. Arterial segments were examined by immunohistochemistry and electron microscopy. Two hours after injury, electron microscopy showed marked decreases in platelet adhesion and neutrophil attachment to the vascular wall surface in FcRgamma-knockout mice compared with wild-type mice. At 7 days after injury, staining with anti-neutrophil antibody showed fewer neutrophils in FcRgamma-knockout mice than in wild-type mice. Computer-aided morphometry performed to measure the neointimal area, intima/media ratio, and stenotic area at 28 days after injury showed a significantly smaller ratio and area in FcRgamma-knockout mice than in wild-type mice (for neointimal area, 16 635 +/- 1406 versus 31 483 +/- 2309 microm2, respectively; for intima/media ratio, 1.25 +/- 0.40 versus 2.68 +/- 0.04, respectively; and for stenotic area, 26.8 +/- 2.1% versus 49.3 +/- 4.1%, respectively). CONCLUSIONS: These results demonstrate that FcRgamma may play important roles in the initiation and generation of neointimal hyperplasia after balloon injury through the activation of platelets by collagen.

Adaptor Proteins, Signal Transducing↗