Mikio Nagashima

Angiogenesis and phenotypic alteration of alveolar capillary endothelium in areas of neoplastic cell spread in primary lung adenocarcinoma.

Normal alveolar capillary endothelium is quiescent in nature and displays anticoagulant thrombomodulin (TM) on its surface. The cytoplasms of these endothelial cells are ultrastructurally non‐fenestrated type, and they barely express von Willebrand factor (vWf). Alveolar fibrosis is accompanied by a capillary endothelium reactive for vWf, and a loss of TM expression. In primary lung adenocarcinoma, neovascularization occurs in association with alveolar fibrosis. In order to study basic factors related to angiogenesis and phenotypic changes of the capillaries located in tumor‐bearing alveolar walls, we examined 37 primary lung adenocarcinomas with electron microscopy and confocal laser scanning microscopy with antibodies for TM, vWf, vascular endothelial growth factor (VEGF), and its receptors (KDR and Flt‐1), and proliferating markers (Ki‐67/proliferating cell nuclear antigen). Tissues microdissected specifically from alveolar walls were used for reverse transcription–polymerase chain reaction (RT–PCR) to assess expressions of mRNA isoforms of VEGF and its receptors. New capillary branching was found by ultrastructural study in the alveolar walls in 12% of the patients. Nuclei of the capillary endothelial cells were reactive for proliferating cell markers. Endothelial fenestrae were developed in 65% of the patients, TM reactivity was lost in the alveolar capillaries, and their cell cytoplasms obtained a reactivity for vWf through a transitional mosaic‐like distribution pattern of both antigens. Besides cytoplasmic VEGF expression in neoplastic cells, tumor‐bearing alveolar walls showed significant expression of mRNA of VEGF165 and KDR. These findings imply that angiogenesis and phenotypic changes of the alveolar capillaries are closely related to a higher expression of tumor‐associated VEGF165 and of KDR in the alveolar walls in primary lung adenocarcinoma.

Increased effect of fucoidan on lipoprotein lipase secretion in adipocytes

AIMS Fucoidan, a sulfated polysaccharide extracted from brown seaweed (F. vesiculosus) is recognized as an effective anticoagulant but its anti-lipidemic potency has not been well defined. We investigated the effect of fucoidan on lipoprotein lipase (LPL) secretion by human adipocytes. MAIN METHODS LPL mRNA and protein expressions were measured using semi-quantitative RT-PCR, ELISA and immunohistochemistry in cultured adipocytes with or without fucoidan treatment. LPL enzyme activity was determined by a fluorometric assay. KEY FINDINGS In cultured adipocytes, fucoidan induced LPL secretion in a dose- and time-dependent manner. An initial increase in LPL was maintained at a significant level but much slower than that in heparin-treated cells. Fucoidan also dose-dependently induced a cofactor of LPL, the apolipoprotein C-II (ApoC-II) secretion. In fucoidan-treated cells, LPL mRNA was time-dependently increased and LPL protein expression was also inceased. Treatment with both heparin and fucoidan showed no further increase in media LPL activity compared to heparin alone. In the conditioned medium from fucoidan-treated cells followed for 4 h, LPL activity decayed exponentially with half-life of about 180 min. In addition, the extracellular LPL mass in cycloheximide (a protein synthesis inhibitor) and fucoidan-treated cells did not change markedly, but LPL shifted significantly from active to inactive form. SIGNIFICANCE These results suggest that fucoidan acts like heparin by releasing LPL in addition to increasing the intracellular transport and decreasing the degradation of LPL in the medium. Furthermore, LPL and ApoC-II secretion induced by fucoidan may be involved in regulating plasma triglyceride lowering clearance.

Fucoidan alleviates high-fat diet-induced dyslipidemia and atherosclerosis in ApoEshl mice deficient in apolipoprotein E expression

Fucoidan, a sulfated polysaccharide extracted from brown seaweeds, possesses many biological activities including anti-inflammatory and antioxidant activities. We aimed to investigate the protective effects of fucoidan on dyslipidemia and atherosclerosis in apolipoprotein E-deficient mice (ApoE(shl) mice) and to elucidate its molecular targets in the liver by using a transcriptomic approach. For 12weeks, ApoE(shl) mice were fed a high-fat diet (HFD) supplemented with either 1% or 5% fucoidan. Fucoidan supplementation significantly reduced tissue weight (liver and white adipose tissue), blood lipid, total cholesterol (TC), triglyceride (TG), non-high-density lipoprotein cholesterol (non-HDL-C) and glucose levels in HFD-fed ApoE(shl) mice but increased plasma lipoprotein lipase (LPL) activity and HDL-C levels. Fucoidan also reduced hepatic steatosis levels (liver size, TC and TG levels, and lipid peroxidation) and increased white adipose tissue LPL activity. DNA microarray analysis and quantitative reverse transcription-polymerase chain reaction demonstrated differential expression of genes encoding proteins involved in lipid metabolism, energy homeostasis and insulin sensitivity, by activating Ppara and inactivating Srebf1. Fucoidan supplementation markedly reduced the thickness of the lipid-rich plaque, lipid peroxidation and foaming macrophage accumulation in the aorta in HFD-fed ApoE(shl) mice. Thus, fucoidan supplementation appears to have anti-dyslipidemic and anti-atherosclerotic effects by inducing LPL activity and inhibiting the effects of inflammation and oxidative stress in HFD-fed ApoE(shl) mice.

Molecular Cytogenetic Characterization of Drug-resistant Leukemia Cell Lines by Comparative Genomic Hybridization and Fluorescence in situ Hybridization

Resistance to chemotherapeutic drugs is one of the major difficulties encountered during cancer chemotherapy. To detect genomic aberrations underlying the acquired drug resistance, we examined three cultured human myelomonocytic leukemia cell sublines each resistant to adriamycin (ADR), 1‐β–1‐d‐arabinofuranosylcytosine (ara‐C), or vincristine (VCR), using comparative genomic hybridization (CGH), fluorescence in situ hybridization (FISH), RT‐PCR, and western blot techniques. Chromosomes 7, 10 and 16 most conspicuously showed frequent aberrations among the resistant sublines as compared to the parental KY–821 cell line. In ADR‐resistant cells, gains at 7q21, 16p12, 16p13.1–13.3, 16q11.1–q12.1, and losses at 7p22–pter, 7q36–qter, 10p12, 10p11.2–pter, 10q21–q25, 10q26–qter were notable. In ara‐C‐resistant cells, no remarkable gain or loss on chromosome 7, but losses at 10p14–pter, 10q26–qter and 16p11.2–p11.3 were observed. In VCR‐resistant cells, gain at 7q21 and losses at 10p11–p13, 10p15 and 16p11.2–p13.3 were found. FISH identified amplified signals for the MDR–1 gene located at 7q21.1 in ADR‐and VCR‐but not ara‐C‐resistant cells, and for the MRP–1 gene located at 16pl3.1 in ADR‐resistant cells. These findings were validated at the mRNA and protein levels. Overlapping of the amplified MRP–1 gene with MDR–1 gene may play a critical part in the acquisition of resistance to ADR. Resistance to ara‐C excluded MDR–1 gene involvement and highlighted other key genes such as MXR gene. Several other genes putatively involved in the development of drug resistance might lie in other aberrated chromosomal regions.

Apolipoprotein E phenotype frequencies in the cerebral infarction.

脳梗塞発症と関連し, 高脂血症は重要なrisk factorのひとつとして, 広く認められているがその詳細はいまだ明らかでない.脳梗塞患者ではHDL中のapoE濃度は対照よりも低下していることが報告され, apoEの動脈硬化における役割が注目されてきている.ApoEはVLDLより分離され, 229個のアミノ酸からなるpolypeptideで遺伝的多形性をもっている.三つの対立遺伝子e2, e3, e4によりcodeされ, apoE2, E3, E4が存在する.これらの組み合わせによりE2/2, E3/2, E3/3, E4/2, E4/3, E4/4の6種類の表現型となる.我々はapo Ephenotypeと脳梗塞との関係を皮質枝梗塞と穿通枝梗塞に分け検討し, また, 血清脂質 (Total cholesterol : T-cho, Triglyceride : T-G, High density lipoprotein cholesterol : HDLcho) 及びapolipoprotein (A-I, A-II, B, C-II, C-III, E) との関連についても検討した.慢性期脳梗塞患者において, 対照と比べ, E3/2が高頻度であった.また, 血清脂質において, 皮質枝梗塞ではT-choの濃度が高い傾向があり, 特にE3/2typeで顕著であった.Apolipoproteinにおいては, 有意な上昇は認めなかった.一方, 穿通枝梗塞においては有意な脂質の上昇は認めなかった.このことはapoEphenotypeの独立の因子としての可能性が示唆される.