Biomedical subjects

Takashi Muramatsu

Publications and source records attributed to Takashi Muramatsu.

At least 19 recordsLinked to original sources

PubMed2007

Midkine as a prognostic marker for gastrointestinal stromal tumors.

PURPOSE: Midkine (MK), a heparin-binding growth factor, has an important role in cancer progression. The outcome of patients with gastrointestinal stromal tumors (GISTs) is correlated with tumor size and mitotic count. The aim of this study was to determine MK expression in GISTs. METHODS: Midkine was detected in 31 (55%) of 57 surgically resected GISTs by immunohistochemistry with a rabbit antibody against MK and peroxidase method. RESULTS: A significant worse outcome of MK-positive patients was found (P < 0.05; log rank test). Multivariate Cox regression analysis showed an independent prognostic impact (relative risk for overall survival 3.64; P < 0.05). Interestingly, MK expression was significantly associated with mitotic rate (P < 0.05; Chi-squared test), but not with tumor size (P = 0.97). CONCLUSIONS: Taken together, MK is a prognostic marker for GIST patients. MK might also be a useful peripheral tumor marker since it can be detected in peripheral serum. Future studies should involve higher GIST patient numbers including tumor and serum samples for detection of MK.

Aged↗

PubMed2006

Midkine as a molecular target: comparison of effects of chondroitin sulfate E and siRNA.

Intraperitoneally administered chondroitin sulfate E inhibited the development of antibody-induced arthritis, a model of rheumatoid arthritis, while chondroitin 4-sulfate showed no effects. Chondroitin sulfate E inhibited in vitro differentiation of osteoclasts, which play key roles in the etiology of rheumatoid arthritis. One of the targets of chondroitin sulfate E is midkine, a heparin-binding growth factor or cytokine. Indeed, a chimeric-type siRNA for midkine inhibited the development of antibody-induced arthritis and adhesion of the omentum to the injured abdominal wall. These results indicate the significance of midkine as a molecular target to treat or prevent rheumatoid arthritis and adhesion after surgery, and the utility of chondroitin sulfate E to inhibit midkine in vivo.

Animals↗

PubMed2006

Midkine plays a protective role against cardiac ischemia/reperfusion injury through a reduction of apoptotic reaction.

BACKGROUND: Midkine (MK) is a heparin-binding growth factor involved in diverse biological phenomena, eg, neural survival, carcinogenesis, and tissue repair. MK could have a protective action against ischemia/reperfusion (I/R) injury in the heart, because MK was shown to have cytoprotective activity in cultured neurons and tumor cells. We investigated this hypothesis in mice with and without genetic MK deletion. METHODS AND RESULTS: Myocardial injury after I/R was produced by transient occlusion of coronary arteries. In wild-type (Mdk+/+) mice, MK expression was increased after I/R in the periinfarct area. Infarct size/area at risk 24 hours after I/R in MK-deficient (Mdk-/-) mice was larger than in Mdk+/+ mice (55.4+/-9.1% versus 32.1+/-5.3%, P<0.05). Terminal dUTP nick end-labeling-positive myocyte population in the periinfarct area in Mdk-/- mice was higher than in Mdk+/+ mice (6.8+/-0.9% versus 3.2+/-0.6%, P<0.05). Left ventricular fractional shortening 24 hours after I/R in Mdk-/- mice was significantly less than that in Mdk+/+ mice (34.3+/-4.4% versus 50.8+/-2.1%, P<0.05). Supplemental application of MK protein to left ventricle of Mdk-/- mice at the time of I/R resulted in reduction of the infarct size. Application of exogenous MK to cultured cardiomyocytes resulted in increased Bcl-2 expression and decreased apoptosis after hypoxia/reoxygenation. CONCLUSIONS: These results suggest that MK plays a protective role against I/R injury, most likely through a prevention of apoptotic reaction. MK is a potentially important new molecular target for treatment of ischemic heart disease.

Animals↗

PubMed2006

Combinational antitumor effect of siRNA against midkine and paclitaxel on growth of human prostate cancer xenografts.

BACKGROUND: Midkine (MK) is a heparin-binding growth factor that promotes the growth, migration, and survival of various cells. Its overexpression has been observed in many human malignancies, making it an attractive therapeutic target. METHODS: We established a human midkine blockade system involving RNA interference. RESULTS: The synthetic small interfering RNA (siRNA) targeting human midkine almost completely inhibited the secretion of midkine by a human prostate cancer cell line, PC-3, and consequently suppressed the proliferation and anchorage-independent growth of the transfected cells. The midkine siRNA, together with atelocollagen, significantly suppressed the growth of PC-3 tumors, which had been subcutaneously xenografted. These inhibitory effects of midkine siRNA were augmented by combinational treatment with a chemotherapeutic, paclitaxel (PTX), both in vitro and in vivo. The dose of PTX (12 mg/kg) used for the combinational treatment did not cause leukopenia or liver damage, which are often reported as serious adverse effects of PTX. The effect of the combination on tumor growth was comparable with that of PTX alone at higher doses that have exhibited severe adverse effects. Midkine siRNA suppressed mainly cell proliferation and slightly angiogenesis, whereas PTX enhanced apoptosis and slightly suppressed angiogenesis. The combination treatment was particularly effective for suppression of angiogenesis; consequently, it had striking effects on all aspects (proliferation, apoptosis, and angiogenesis). CONCLUSIONS: We established a novel and safe cancer therapy strategy involving midkine siRNA combined with PTX, with less adverse effects.

Animals↗

PubMed2006

Neuroglycan C is a novel midkine receptor involved in process elongation of oligodendroglial precursor-like cells.

Midkine is a heparin-binding growth factor that promotes cell attachment and process extension in undifferentiated bipolar CG-4 cells, an oligodendroglial precursor cell line. We found that CG-4 cells expressed a non-proteoglycan form of neuroglycan C, known as a part-time transmembrane proteoglycan. We demonstrated that neuroglycan C before or after chondroitinase ABC treatment bound to a midkine affinity column. Neuroglycan C lacking chondroitin sulfate chains was eluted with 0.5 m NaCl as a major fraction from the column. We confirmed that CG-4 cells expressed two isoforms of neuroglycan C, I, and III, by isolating cDNA. Among three functional domains of the extracellular part of neuroglycan C, the chondroitin sulfate attachment domain and acidic amino acid cluster box domain showed affinity for midkine, but the epidermal growth factor domain did not. Furthermore, cell surface neuroglycan C could be cross-linked with soluble midkine. Process extension on midkine-coated dishes was inhibited by either a monoclonal anti-neuroglycan C antibody C1 or a glutathione S-transferase-neuroglycan C fusion protein. Finally, stable transfectants of B104 neuroblastoma cells overexpressing neuroglycan C-I or neuroglycan C-III attached to the midkine substrate, spread well, and gave rise to cytoskeletal changes. Based on these results, we conclude that neuroglycan C is a novel component of midkine receptors involved in process elongation.

Animals↗

PubMed2006

N-Acetylglucosamine 6-O-sulfotransferase-1 is required for brain keratan sulfate biosynthesis and glial scar formation after brain injury.

Keratan sulfate (KS) is a glycosaminoglycan composed of repeating disaccharide units with sulfate residues at the C6 positions of galactose and N-acetylglucosamine (GlcNAc). The N-acetylglucosamine 6-O-sulfotransferase(s) (GlcNAc6ST) involved in the synthesis of KS in the central nervous system (CNS) has long been unidentified. Here, we report that a deficiency of GlcNAc6ST-1 leads to loss of 5D4-reactive brain KS and reduction of glial scar formation after cortical stab injury in mice. During the development of mice deficient in GlcNAc6ST-1, KS expression in the brain was barely detectable with the KS-specific antibody 5D4. The reactivity of 5D4 antibody with protein tyrosine phosphatase zeta (PTPzeta), a KS proteoglycan (KSPG), was abolished in the deficient mice. In adults, brain injury induced 5D4-reactive KS synthesis in the wounded area in wild-type (WT) mice but not in the deficient mice. Glial scar is formed via the accumulation of reactive astrocytes and is a major obstacle to axonal regeneration by injured neurons. Reactive astrocytes appeared to similar extents in the two genotypes, but they accumulated in the wounded area to a lesser extent in the deficient mice. Consequently, the deficient mice exhibited a marked reduction of scarring and enhanced neuronal regeneration after brain injury. These findings highlight the indispensable role of GlcNAc6ST-1 in brain KS biosynthesis and glial scar formation after brain injury.

Acetylglucosamine↗

PubMed2006

Mice doubly deficient in the midkine and pleiotrophin genes exhibit deficits in the expression of beta-tectorin gene and in auditory response.

alpha-Tectorin and beta-tectorin are major noncollagenous proteins of the tectorial membrane, which plays a crucial role in the reception of sonic signals in the cochlea. Midkine and pleiotrophin are closely related proteins that serve as growth factors and cytokines. In mice doubly deficient in the midkine gene and pleiotrophin gene, expression of beta-tectorin mRNA was nearly abolished in the cochlea on day 1 and 7 after birth. Expression of alpha-tectorin mRNA was unaffected in the double knockout mice, and expression of beta-tectorin mRNA was not altered in mice deficient in only the midkine or pleiotrophin gene. In newborn wild-type mice, both midkine and pleiotrophin were expressed in the greater epithelial ridge of the cochlea, in which beta-tectorin mRNA was strongly expressed. These results indicate that either midkine or pleiotrophin is required for significant expression of beta-tectorin. In agreement with the view that beta-tectorin is essential for normal auditory function, mice doubly deficient in both midkine and pleiotrophin genes exhibited very severe auditory deficits. We observed that mice deficient in either midkine or pleiotrophin gene were also impaired in their auditory response, but the level of the deficit was generally low or moderate. The present finding illustrates the importance of growth factor expression in the cochlea for auditory function.

Animals↗

PubMed2006

Doxorubicin-conjugated anti-midkine monoclonal antibody as a potential anti-tumor drug.

BACKGROUND: Midkine is a heparin-binding growth factor preferentially expressed in tumor cells. The present study was performed to utilize anti-midkine antibody for tumor therapy. METHODS: A monoclonal antibody to midkine was raised by immunizing mice deficient in the midkine gene. The binding site of the antibody was studied by using N-terminal half and C-terminal half of midkine, both of which were chemically synthesized. Doxorubicin (DOX)-conjugate of the antibody was produced by chemical conjugation. The effects of the antibody and the conjugate on cell growth were examined using a midkine-secreting tumor cell, i.e. human hepatocellular carcinoma cell (HepG2). RESULTS: The monoclonal antibody bound to the N-terminal half of midkine. The antibody did not inhibit the growth of HepG2 cells probably because the active domain of midkine is in the C-terminal half. We produced the antibody conjugated with DOX with the hope that the conjugate would be internalized accompanied with midkine. Indeed, the antibody-DOX conjugate significantly inhibited the growth of HepG2 cells compared with DOX-conjugated control IgG. CONCLUSION: The result raises the possibility of using anti-midkine antibody conjugated with DOX for cancer therapy.

Antibiotics, Antineoplastic↗

PubMed2006

Midkine, a heparin-binding growth factor, produced by the host enhances metastasis of Lewis lung carcinoma cells.

Midkine is a heparin-binding growth factor and is expressed by a number of tumor cells, contributing to their growth both in vitro and in vivo. Spontaneous lung metastasis of Lewis lung carcinoma cells, which did not significantly express MK, was significantly less extensive in mice deficient in the midkine gene than in wild-type mice, when the tumor was subcutaneously grown above the thigh. Midkine strongly enhanced migration of Lewis lung carcinoma cells in vitro. Therefore, midkine is also a host factor enhancing tumor metastasis. Anti-midkine therapy for malignancy may act on midkine produced by both the tumor and host.

Animals↗

PubMed2006

Controlled release of small interfering RNA targeting midkine attenuates intimal hyperplasia in vein grafts.

OBJECTIVE: Intimal hyperplasia is a major obstacle to patency after vein grafting. Despite of a diverse array of trials to prevent it, a satisfactory therapeutic strategy for clinical use has not been established. However, sufficient inhibition of early stages of intimal hyperplasia may prevent this long-term progressive disease. Midkine (MK) is a heparin-binding growth factor that was originally discovered as the product of a retinoic acid-responsive gene. We previously demonstrated that MK-deficient mice exhibit a striking reduction of neointima formation in a restenosis model, which is reversed on systemic MK administration. In this study, we evaluated a strategy of using small interfering RNA (siRNA) targeting MK as a therapy for vein graft failure. METHODS: We first made a highly effective siRNA to rabbit MK. Jugular vein-to-carotid artery interposition vein grafts, which are applied to a low flow condition, were made in Japanese white rabbits. Small interfering RNA mixed with atelocollagen was administrated to the external wall of grafted veins. Cy3-conjugated stabilized siRNA was used to confirm its stability and successful transfer into the vein graft wall. Neointimal hyperplasia was evaluated 4 weeks after the operation. The proliferation index and leukocyte infiltration were determined. RESULTS: MK expression was induced and reached the maximum level 7 days after operation. Fluorescence of Cy3-labeled siRNA could be detected in the graft wall even 7 days after operation. Knockdown of the gradually increasing expression was achieved by perivascular application of siRNA using atelocollagen. The intima-media ratio and the intima thickness at 28 days after grafting were both reduced >90% by this treatment compared with controls. This phenomenon was preceded by significant reductions of inflammatory cell recruitment to the vessel walls and subsequent cell proliferation in MK siRNA-treated grafts. CONCLUSIONS: These results suggest that midkine is a candidate molecular target for preventing vein graft failure. Furthermore, for clinical applications of siRNA, a single intraoperative atelocollagen-based nonviral delivery method could be a reliable approach to achieve maximal function of siRNA in vivo. This strategy may be a useful and practical form of gene therapy against human vein graft failure.

Animals↗

PubMed2006

Determination of substrate specificity of sulfotransferases and glycosyltransferases (proteoglycans).

Proteoglycans have sulfated linear polysaccharide chains, that is, heparan sulfate, heparin, chondroitin sulfates, dermatan sulfate, and keratan sulfate. Many glycosyltransferases and sulfotransferases are involved in biosynthesis of the polysaccharides. Specificities of these enzymes have been mainly determined by evaluating their activities to various acceptor carbohydrates and by analyzing the structure of the products. For the latter purpose, enzymatic hydrolysis using heparitinases, heparinase, and chondroitinases or chemical degradation employing nitrous acid deamination has been effectively used in combination with high-performance liquid chromatography (HPLC) of the degraded products. As examples, we describe methods for assays and product characterization of sulfotransferases involved in biosynthesis of these polysaccharides, namely heparan sulfate 2-sulfotransferase, heparan sulfate 6-sulfotransferases, chondroitin 4-sulfotransferases, chondroitin 6-sulfotransferase, N-acetylgalactosamine 4-sulfate 6-sulfotransferase, and N-acetylglucosamine 6-sulfotransferases.

Deamination↗

PubMed2006

Identification of proteoglycan-binding proteins.

There are many proteins that bind to proteoglycans; they include proteins in extracellular matrices, growth factors or cytokines, plasma proteins, transmembrane proteins, and cytoplasmic proteins as listed in this chapter. Proteins that bind to a proteoglycan have been searched by using a proteoglycan as a ligand. Alternatively, a ligand protein has been used to find a proteoglycan as a binding partner. When the glycosaminoglycan (GAG) portion of a proteoglycan is responsible for the binding, a native proteoglycan is necessary for the analysis of binding. When the protein portion is responsible for the binding, a recombinant core protein without GAG chains may be used for analysis. This chapter describes experimental procedures dealing with two native proteoglycans, versican (PG-M) and syndecan-4 (ryudocan). Versican has been identified as a proteoglycan with binding capability to a growth factor, midkine. Purified syndecan-4 has been used to identify proteins that bind to the proteoglycan.

Animals↗

PubMed2006

Female infertility in mice deficient in midkine and pleiotrophin, which form a distinct family of growth factors.

Midkine and pleiotrophin form a family of growth factors. Mice deficient in one of the genes show few abnormalities on reproduction and development. To understand their roles in these processes, we produced mice deficient in both genes; the double deficient mice were born in only one third the number expected by Mendelian segregation and 4 weeks after birth weighed about half as much as wild-type mice. Most of the female double deficient mice were infertile. In these mice, the numbers of mature follicles and of ova at ovulation were reduced compared to numbers in wild-type mice. Both midkine and pleiotrophin were expressed in the follicular epithelium and granulosa cells of the ovary. The expression of these factors in the uterus was dramatically altered during the estrous cycle. The diestrus and proestrus periods were long and the estrus period was short in the double deficient mice, indicating the role of the factors in the estrous cycle. Furthermore, vaginal abnormality was found in about half of the double deficient mice. These abnormalities in combination resulted in female infertility. Therefore, midkine and pleiotrophin, together with their signaling receptors, play important roles in the female reproductive system.

Animals↗

PubMed2006

Midkine, a heparin-binding growth factor, is expressed in neural precursor cells and promotes their growth.

Midkine, a heparin-binding growth factor, was found to be expressed in neural precursor cells, which consist of neural stem cells and the progenitor cells. When embryonic brain cells were allowed to form neurospheres enriched in neural precursor cells, numbers were significantly smaller from the midkine-deficient brain than from the wild-type brain. Dissociated neurosphere cells yielded nestin-positive neural precursor cells and differentiated neuronal cells upon culture on a substratum. Neural precursor cells from the midkine-deficient brain spread poorly and grew less effectively on a substratum coated with poly-l-lysine than the cells on midkine-coated substratum. Neural precursor cells from the wild-type brain spread and grew well on both the substrata. Differentiation to neurons and glia cells was not affected by the absence of midkine. Heparitinase digestion of dissociated neurosphere cells resulted in poor growth of neural precursor cells, while chondroitinase digestion had no effect. These results indicate that midkine is involved in the growth of neural precursor cells and suggest that the interaction with heparan sulfate proteoglycans is important in midkine action to these cells.

Animals↗

PubMed2006

Differential localization of laminin gamma and integrin beta in primary cultures of the rat gingival epithelium.

OBJECTIVES: The aim of this study was to investigate the differential immunolocalization of laminin gamma(2) and integrin beta(4) in primary cultures of the rat gingival epithelium. METHODS: The gingival epithelium was obtained from Sprague-Dawley rats and was cultured in serum-free keratinocyte growth medium (DK-SFM). Western blotting analysis, immunofluorescence, confocal laser scanning microscopy (CLSM), and immuno-gold labeling for laminin gamma(2) and integrin beta(4) were employed. CLSM images for laminin and integrin were analyzed in horizontal (x-y axis) and in vertical (x-z axis) sections. RESULTS: Both laminin gamma(2) and integrin beta(4) were detected by Western blot analysis in the gingival epithelium. Immunolocalization of laminin gamma(2) was distinct in the cytoplasm to form one or two irregular rings in gingival epithelial cells. By contrast, integrin beta(4) was localized diffusely in the cytoplasm. F-actin (indicating actin filaments) was clearly discernible at the periphery of the cytoplasm to form a cellular fringe. In x-z axis images obtained by CLSM, laminin gamma(2) was recognized as large foci in the most inner portion just above the basal plasma membrane. Integrin beta(4) existed in the area where F-actin was labeled surrounding the membrane. Immuno-electron microscopy showed that 10nm colloidal gold particles indicating laminin gamma(2) were mainly localized at the extracellular portion and in the peripheral cytoplasm, whereas integrin beta(4) was distributed in the cytoplasm close to the basal plasma membrane but not in extracellular regions. CONCLUSIONS: In primary cultures of the rat gingival epithelium, both laminin gamma(2) and integrin beta(4) may be produced by the epithelium, and irregular rings of laminin gamma(2) are formed in areas where gingival cells adhere to the extracellular matrix.

Actins↗

PubMed2006

The expression and localization of osteopontin in the mouse major salivary glands.

The present study investigated the expression and distribution of osteopontin in the mouse major salivary glands. The level of osteopontin expression in the mouse submandibular gland was higher (12.7-fold) than that in parotid and sublingual glands at the mRNA level. By Western blot analysis, intense positive bands were seen at the predicted molecular mass (about 55 kDa) in all the major salivary glands, while an approximately 30 kDa band of osteopontin was detected only in the submandibular gland. Indirect immunofluorescent and immuno-electron microscopy analyses demonstrated the localization of osteopontin in the luminal (apical) membranes of acinar cells in all the salivary glands. Osteopontin was also localized at the lumen of acini in the submandibular gland. These results suggest that the expression of osteopontin in the submandibular gland is different from that in the parotid and sublingual glands and that osteopontin may be degraded in the mouse submandibular gland.

Animals↗

PubMed2006

Growth factor midkine is involved in the pathogenesis of diabetic nephropathy.

Diabetic nephropathy is a life-threatening disease associated with diabetes mellitus. Longstanding hyperglycemia induces pathological reactions of glomerular mesangial cells, such as overproduction of extracellular matrix, which finally lead to nephropathy. However, the mechanisms underlying its pathogenesis have not been completely elucidated. Using the Streptozotocin-induced model of diabetes, we report that mice deficient in the growth factor midkine (Mdk-/-) exhibited strikingly milder nephropathy than Mdk+/+ mice, even though both mice showed similar extents of hyperglycemia after Streptozotocin injection. Midkine expression was induced in the glomerular mesangium of Mdk+/+ mice with diabetic nephropathy and in primary cultured mesangial cells exposed to high glucose. Mdk-/- mesangial cells exhibited reduced phosphorylation of protein kinase C and extracellular signal-regulated kinase as well as reduced production of transforming growth factor-beta(1) on high glucose loading. Addition of exogenous midkine restored extracellular signal-regulated kinase phosphorylation in Mdk-/- cells under high glucose conditions, whereas a midkine antisense oligodeoxynucleotide suppressed midkine in Mdk+/+ cells. Therefore, this study identifies midkine as a key molecule in diabetic nephropathy and suggests that midkine accelerates the intracellular signaling network evoked by hyperglycemia in nephropathy.

Animals↗

PubMed2006

Connexin 43 transfection in basaloid squamous cell carcinoma cells.

To investigate the relationship between the expression of connexin in basaloid squamous cell carcinomas (BSCC) and their rapid proliferation and invasive potential, we examined the effect of overexpression of connexin 43 (Cx43) in a BSCC-derived cell line (BSC-OF). BSC-OF was transfected with Cx43 to obtain 15 clones with a stable expression of Cx43. In these cells, although Cx43 was distributed throughout the cytoplasm, it did not form connexon plaque. In almost all of the clones, cell proliferation was clearly suppressed. Furthermore, we investigated cell migration and invasion in three clones that showed a remarkable down-regulation in cell growth, and found that Cx43 transfection showed no significant effect on either. These results suggest that Cx43 plays a role as a tumor suppressor in the cytoplasm of Cx43-transfected BSC-OF cells. However, no definite correlation was found between Cx43 and cell migration and invasion.

Carcinoma, Squamous Cell↗