9 Stephan G, Xi XP, Yasuko K, Hiroaki K, Eckart F. TNF-{alpha}–Induced Migration of Vascular Smooth

合集下载

金泽咲希

金泽咲希

金泽咲希(日本籍)原名满丽曾,1976年出生,16岁加入国家队。

1995年参加世界乒乓球锦标赛,在1998年加入日本国籍并改名为金泽咲希,2006年代表日本出征第48届不莱梅世界锦标赛,并为日本队夺得团体铜牌,作为日本少有的左撇子直拍选手活跃在日本乒坛,于2009年2月隐退。

5、小山修加(日本籍)王娇在2002年归化日本后改名为小山修加,在2006年的国际排练女排[微博]大奖赛中替日本队出征,其出色的弹跳和高点进攻频频得分,吸引了所有观众的们目光,2007年又被日本队教练列入了12人的主力名单。

后于2010年退役。

6、张金玲(韩国籍)前中国国家射击队队员张金玲2006年5月与韩国人金大庆(音译)结婚,2009年加入韩国籍。

2009年6月10日,张金玲夺得了韩国第五届警护处技艺全国射击大赛女子组50米步枪三姿个人和团体冠军。

7、高堰雪梅(日本籍)曾代表中国队出征巴塞罗那奥运会的女子体操运动员和雪梅在1995年赴日留学,2006年获得日本国籍改名为高堰雪梅。

在2007年的日本体操锦标赛中,30岁“高龄”的高堰雪梅力压10多岁的小运动员们,拿下了平衡木的单项冠军。

8、苏文燕(计划入韩国籍)今年2月18日,有媒体报道称中国女篮国手徐春梅15岁的女儿苏文燕有计划加入韩国,成为韩国新一代篮球明星。

据韩国方面表示,苏文燕成为职业篮球选手之前,俱乐部方面将负担她在韩国的教育费和生活费等一切费用,同时还将帮助她获得韩国国籍。

从归化运动员的数据中我们不难发现,这些运动员归化的时间都集中在90年代末21世纪初这样一个时间段,主要集中在乒乓球、羽毛球[微博]、体操、篮球等中国传统优势项目上。

经过结合当时的现状,发现其中原因有四:1、这一时间段正是中国的社会与经济逐渐开放的年代,中国运动员们也更多地通过各种渠道认识到和日本、韩国运动员的收入水平与训练水平的巨大差距。

选择更好的生活、训练条件是促使运动员们出走的原因之一。

2、中国在此类传统优势项目上不乏优秀高水平运动员,但是奥运会、亚运会、世锦赛等世界级大赛的参赛名额有限,这必然导致部分运动员没有机会代表国家参加世界级大赛。

Kamikaze Girls系列

Kamikaze Girls系列

Kamikaze Premium系列Kamikaze Girls系列Kamikaze Street系列2009-08-15 17:18Kamikaze Premium系列Kamikaze Premium Vol.01 矢藤あきKamikaze Premium Vol.02 木村那美Kamikaze Premium Vol.03 白石麻梨子Kamikaze Premium Vol.04 星乃まりんKamikaze Premium Vol.05 小町ななみKamikaze Premium Vol.06 今野由愛Kamikaze Premium Vol.07 和希結衣Kamikaze Premium Vol.08 姫野愛Kamikaze Premium Vol.09 田中美久Kamikaze Premium Vol.10 @YOUKamikaze Premium Vol.11 白鳥あきらKamikaze Premium Vol.12 吉野サリーKamikaze Premium Vol.13 鈴木麻奈美Kamikaze Premium Vol.14 Jun NadaKamikaze Premium Vol.15 日夏ともえKamikaze Premium Vol.16 佐仓Kamikaze Premium Vol.17 山崎亜美Kamikaze Premium Vol.18 佐佐木渚沙Kamikaze Premium Vol.19 片瀬久留美Kamikaze Premium Vol.20 桃瀬ゆうなKamikaze Premium Vol.21 藤崎怜里Kamikaze Premium Vol.22 光咲玲奈Kamikaze Premium Vol.23 NatsumiKamikaze Premium vol.24 新堂有望(Yumi Shindo) (KP-024) Kamikaze Premium Vol.25 榊彩弥(Aya Sakaki) (KP-025) Kamikaze Premium Vol.26 いおり(Iori) (KP-026)Kamikaze Premium Vol.27 夏樹唯(Yui Natsuki) (KP-027) Kamikaze Premium Vol.28 Black Jesus 姫野爱瀬咲るな夏樹唯Kamikaze Premium Vol.29 宝月ひかるKamikaze Premium Vol.30 中野美奈Kamikaze Premium Vol.31 夏樹唯Kamikaze Premium Vol.32 Azusa Isshiki 一色あずさKamikaze Premium Vol.33 姬野愛Kamikaze Premium Vol.34 Kumi MizusawaKamikaze Premium Vol.35 姬野愛Kamikaze Premium Vol.36 Ai Himeno 姫野愛Kamikaze Premium Vol.37 姬川Kamikaze Premium Vol.38 川愛加奈Kamikaze Premium Vol.39 姫野愛Kamikaze Premium Vol.40 Ami Matsuda 松田亜美Kamikaze Premium Vol.41 Arisa Suzufusa 鈴房Kamikaze Premium Vol.42 Hitomi Nakagawa 中川瞳Kamikaze Premium Vol.43Kamikaze Premium Vol.44Kamikaze Premium Vol.45Kamikaze Premium Vol.46Kamikaze Premium Vol.47Kamikaze Premium Vol.48Kamikaze Premium Vol.49Kamikaze Girls vol.01 水原みなみKamikaze Girls vol.02 姫野愛Kamikaze Girls vol.03 稻葉夕輝Kamikaze Girls vol.04 RinKamikaze Girls vol.05 大迫ゆみKamikaze Girls vol.06 沢井真帆Kamikaze Girls vol.07 佐々木渚沙Kamikaze Girls vol.08 上原美紀Kamikaze Girls vol.09 華美月Kamikaze Girls vol.10 さくらのKamikaze Girls vol.11 白鳥あきらKamikaze Girls vol.12 咲月美羽Kamikaze Girls vol.13 水無月なぎさKamikaze Girls Vol.14 葉山くみこKamikaze Girls Vol.15 夏樹唯Kamikaze Girls Vol.16 葉山リカKamikaze Girls Vol.17 滝沢优季Kamikaze Girls vol.18 大城舞衣子Kamikaze Girls Vol.19 加藤まみKamikaze Girls Vol.20 铃木えみりKamikaze Girls Vol.21 酒菜いるかKamikaze Girls Vol.22 瀬咲るなKamikaze Girls Vol.23 枫はるかKamikaze Girls Vol.24 友恵ゆいKamikaze Girls Vol.25 Hikaru Houzuki 寶月ひかるKamikaze Girls Vol.26 今井みすずKamikaze Girls Vol.27 S級美女一色あずさKamikaze Girls Vol.28 松藤美貴Kamikaze Girls Vol.29 Arisa Suzufusa 鈴房Kamikaze Girls Vol.30 中野美奈Kamikaze Girls Vol.31 川野優Kamikaze Girls Vol.32 Yukari Mayama 真山ゆかり. Kamikaze Girls Vol.33 Kana Kawai 川愛加奈Kamikaze Girls Vol.34 中川瞳Kamikaze Girls Vol.35 上原のぞみKamikaze Girls Vol.36 下真纪Kamikaze Girls Vol.37 園原Kamikaze Girls Vol.38 神谷りのKamikaze Girls Vol.39 長澤リオン超诱惑美臀Kamikaze Girls Vol.40 Chihiro Asakura 朝倉ちひろKamikaze Girls Vol.41Kamikaze Girls Vol.42 野々宮りんKamikaze Girls Vol.43Kamikaze Girls Vol.44Kamikaze Street Vol.01 今野麻衣子Maiko Imano Kamikaze Street Vol.02Kamikaze Street Vol.03 Iwaki Saori 岩城沙織Kamikaze Street Vol.04 AkiKamikaze Street Vol.05Kamikaze Street Vol.06Kamikaze Street Vol.07Kamikaze Street Vol.08 熊田ありさKamikaze Street Vol.09 瀬名えみりKamikaze Street Vol.10 浅倉りほKamikaze Street Vol.11Kamikaze Street Vol.12 桜井Kamikaze Street Vol.13 裡山加與Kayo Satoyama Kamikaze Street Vol.14Kamikaze Street Vol.15 小倉美穂Kamikaze Street Vol.16 榎本らん, 愛Kamikaze Street Vol.17Kamikaze Street Vol.18 相川純菜。

Following each of the pixel of that image

Following each of the pixel of that image

专利名称:Following each of the pixel of that image 发明人:松居 美佳,中井 賢,▲よし▼井 健人,森田 克之,宮部義幸申请号:JP1996040489申请日:19960202公开号:JP3673002B2公开日:20050720专利内容由知识产权出版社提供摘要:PROBLEM TO BE SOLVED: To provide a multi-level framing data generation device and a method capable of easily performing framing by using multilevel data and outputting beautiful multi-level framing data corresponding to a typeface and an output device. SOLUTION: By this multi-level framing data generation device provided with a multi-level data inversion means 2 for inverting the respective pixels of a picture in the multi-level data for forming the picture constituted from three or more multiple levels corresponding to a prescribed inversion rule and generating multi-level inversion data, a thick width data generation means 3 for sending out thick width data obtained by using the multi-level data and an inversion data subscribing means 4 for subscribing a part of the inversion data to the thick width data and generating the multi-level framing data, the beautiful multi-level framing data are easily generated by using the multi-level data.申请人:松下電器産業株式会社地址:大阪府門真市大字門真1006番地国籍:JP代理人:松田 正道更多信息请下载全文后查看。

赛车皇后相马茜

赛车皇后相马茜

赛车皇后相马茜相马茜 1998年首度参加赛车队RACE QUEEN,她甜美的外型让演艺圈与赛车界为之惊艳。

从2000-2002年陆续参加XANAVI 与YeLLOW CORN车队的RQ,优异的表现与其亮丽的外形,让她赢得日本第一的知名度与人气。

为了让自己有更全方位的发展,相马茜从YeLLOW CORN 引退後正式以一位艺人的身份,以TV为中心,进军演艺圈。

中文名:相马茜外文名:Akane Souma国籍:日本出生地:日本福冈县出生日期:1975年9月8日职业:艺人写真集Career2002 Yellow Corn Gal写真集“史上最强RQ Yellow Corn发卖相马茜1st写真集“e.girl”2nd.写真集”PASSION FRUITS”发卖中DVD1st.DVD“PASSION FRUITS”2nd.DVD“Ruddy““HOLD UP”3rd.DVD“Love Prisoner”发卖中1st.DVD&VIDEO『DELICIOUS CAPRICIOUS』2nd『Delicious Capricious&more 』相马茜篇DVD&VIDEO『So Much in Love』发卖中活动经历代言侬特利之食品广告、海报、活动演出TVANB『T2×SHOW』TX『爆NEW』『HOTDOG PRESS』『C ool Girls』等演出CF1999 KIRIN JUG’S「日立速乾名人」「日经娱乐」「东海隐形眼镜」「万叶之汤」2004年5月预定发行唱片2004年3月预定拍摄台湾偶像剧2004年3月在线游戏年度代言人2004年1月国际书展签握会2004年GOLDEN CARIPE代言人2003~2004年台湾地区车展代言人2002年Yellow Corn RQ (Race Queen)1999~2001年Xanavi RQ (Race Queen)1998年Kosei RQ杂志『CR+』『cooee(封面)』『写真logo(封面)』『Playng2(封面)『艺能NIPPON(封面)』『FRIDAY』『FLASH』『FLASH-EX』『周刊PLAYBOY』花边座右铭:经常不能忘记要有感谢的心擅长的语言:日语、福冈方言觉得自己最美的地方:肚脐觉得自己最丑的地方:大腿觉得自己个性上最大的优点:不论在哪里都能睡觉最爱的人:母亲和祖母初恋的年龄:5岁希望几岁结婚:不太想结婚最爱收藏的东西:帽子最大的愿望:父母亲的幸福最害怕的东西:幽灵最讨厌哪种类型的人:不爱清洁的人最尴尬的事情:被自行车的後轮扯到裙子、裙子掉下来时最喜欢做的事情:买东西最爱的书:流行杂志最喜欢的作家:没有特别喜欢哪一位最爱的明星:木尾芽衣子、元斌(韩国人)最爱的电影:醉拳最爱的歌:宇多田光的歌曲、彩虹乐团的歌曲最爱吃的食物:乌鱼子、生牛肝(生吃)最喜欢的颜色:红色最喜欢的运动:篮球最喜欢的植物:樱花最想旅行的地方:想到台湾一些不曾去过的地方最怕的动物(昆虫):蜘蛛难过的时候最想做的事情:一直哭高兴的时候最想做的事情:给双亲打电话当生命到了最后一天最想做的事情:向曾经帮过我的人致谢参演偶像剧2009民视《终极三国》饰校长(王允)妻、貂蝉养母。

爱の深さは膝くらい(小西克幸X下野紘、前野智昭)特典CD附文库

爱の深さは膝くらい(小西克幸X下野紘、前野智昭)特典CD附文库

【12/25新作在線翻譯】愛の深さは膝くらい(小西克幸X下野紘、前野智昭)特典CD附発売元インターコミュニケーションズ原作依田沙江美(芳文社花音コミックス)キャスト下野紘(坂下昴)小西克幸(石倉正規)前野智昭(川田時成)TRACK I石仓正规:我是石仓正规,二十八岁,为了继承神社而回到了老家,本打算暂时享受一下悠哉游哉的状态的,不过在父亲的交涉下,被介绍了个代课老师的工作,从九月开始,我完全成了高中教师,到了第二年的现在,已经很适应这一工作场合,学生们也很亲近我,就我来说,过的非常愉快,但是……真由子:是啊是啊,石仓老师有女朋友吗?石仓正规:如果我说没有的话,你会和我交往吗?真由子:讨厌,老师,这样你会被抓走哦。

石仓正规:是啊,所以你们就别问这种问题了。

哎呀!有页讲义掉下去了呢。

啊,喂,不要用脚踩住它啊。

(哇,是坂下昴)给你添麻烦了,多亏了你这页讲义才没有被风吹走。

坂下昴:不用谢。

石仓正规:被狠狠的……踩了?!真由子:老师,你一个人住?石仓正规:要是这样的话,你就过来帮我打扫?小林奈绪:我去吧?石仓正规:(啊~坂下的冰冷视线好可怕!)坂下昴:老师。

石仓正规:什么事?坂下昴:啊!对不起,我太不小心了。

真由子:老师,你没事吧?坂下,你都做了什么啊!小林奈绪:老师,快把毛衣脱掉石仓正规:(再怎么说也太过分了吧?我果然被坂下讨厌了吧?不过,一般会把砚台里的墨汁往别人升上泼的吗?就这样想来想去的迎来了周末,我被警察叫了过去,正确的说是被将我的名字交给了派出所的坂下叫了过去。

)石仓正规:你好,我是接到电话过来的石仓。

警察:哦,辛苦了,你是他们的班主任吗?石仓正规:啊,不是的,我是……坂下昴:老师!石仓正规:啊坂下昴:对不起,我身边只有社团的联络本川田时成:昴……石仓正规:哦,经常和坂下在一起的家伙,好像是叫川田时成吧,他也在一起啊。

坂下昴:我和川田都跟着石仓老师在书法部学习石仓正规:诶?川田……坂下昴:老师,你不要糊涂了,虽然他只是旁观的,不过也算是部员吧石仓正规:啊……是……抱歉……是这样吗?警察:这位老师还真是年轻啊石仓正规:啊,我虽说也是老师,不过也是最近才临时当上的坂下昴:老师是日吉【音译】神社的石仓神官警察:诶?是日吉神社的石仓神官?那你是宫司的儿子?【注:宫司是指神社的最高神官】石仓正规:是的警察:哎呀呀,平常总是受你父亲照顾石仓正规:(老爸的名字总是这么管用啊,虽然这样是很方便没错)那个……请问他们两个做了什么?警察:我接到通知说有人在河床那边打架,等我们到达那边的时候就只剩下他们两个了,所以就暂且先带他们回派出所问话了。

FLEXIBLE TUBE OF ENDOSCOPE

FLEXIBLE TUBE OF ENDOSCOPE

专利名称:FLEXIBLE TUBE OF ENDOSCOPE发明人:KASAI TADASHI,葛西 忠志,IWASAKA KIKUO,岩坂 喜久男,NANBA HIDEO,難波 英男,HAYAKAWA SHINJI,早川 真司申请号:JP特願平11-121900申请日:19990428公开号:JP特開2000-308615(P2000-308615A)A公开日:20001107专利内容由知识产权出版社提供专利附图:摘要:PROBLEM TO BE SOLVED: To provide a flexible tube of endoscope excellent in peeling strength of an outer skin, causing a buckling little, easy to bend and excellent in insertability and operability. SOLUTION: In the flexible tube of endoscope in which a coating 21b formed of a thermoplastic synthetic resin is applied to at least one ofstrands 21a forming a strand bundle of a network-like tube, and the coating 21b part ismelted and welded to an outer skin 30, the coating 21b applied to at least one of the strands 21a of the network-like tube is welded from a welded portion to the outer skin 30 to the position in the middle of the adjacent surface with adjacent strands with respect to its adjacent strand 21a.申请人:ASAHI OPTICAL CO LTD,旭光学工業株式会社地址:東京都板橋区前野町2丁目36番9号国籍:JP代理人:三井 和彦更多信息请下载全文后查看。

GENE ENCODING PROMOTER DOMAIN OF TUMOR SUPPRESSOR

GENE ENCODING PROMOTER DOMAIN OF TUMOR SUPPRESSOR

专利名称:GENE ENCODING PROMOTER DOMAIN OF TUMOR SUPPRESSOR GENE P51 AND USETHEREOF发明人:SAKAI, Toshiyuki,KAGAYA, Shigehide,SATO, Takamichi,SUKENAGA, Yoshikazu,FUJII, Hideji 申请号:JP2000004261申请日:20000628公开号:WO01/000818P1公开日:20010104专利内容由知识产权出版社提供摘要:A gene encoding the promoter domain of a protein p51 which is capable of inducing cell death; and a gene encoding the 5'-nontranslation domain of p51. These genes are useful in diagnosing and treating diseases typified by cancer caused by abnormal regulation of cell proliferation. Antisense DNAs and antisense RNAs of these genes; nucleic acid probes comprising the full sequences of these genes or parts thereof;a method for detecting the above genes or genes analogous thereto by using these nucleic acid probes; transformants having the above genes transferred thereinto; a method for screening a drug by using the same; and the like. These genes are also useful in diagnosing and treating diseases such as cancer.申请人:SAKAI, Toshiyuki,KAGAYA, Shigehide,SATO, Takamichi,SUKENAGA, Yoshikazu,FUJII, Hideji地址:28-1, Matsugasaki Kowakicho Sakyo-ku Kyoto-shi Kyoto 606-0957 JP,11-2, Fujimi 1-chome Chiyoda-ku Tokyo 102-8172 JP,2-336, Kitabukurocho Omiya-shi Saitama 330-0835 JP,6-8-22, Kamiochiai Yono-shi Saitama 338-0001 JP,2-21-15-102, Ebaracho Nakano-ku Tokyo 165-0023 JP,3-48-21-1201, Honmachi Shibuya-ku Tokyo 151-0071 JP 国籍:JP,JP,JP,JP,JP,JP代理机构:ASAMURA, Kiyoshi更多信息请下载全文后查看。

Deodorants, deodorant sheets, filter sheets and fu

Deodorants, deodorant sheets, filter sheets and fu

专利名称:Deodorants, deodorant sheets, filter sheets and functional papers as well as filteringmediums for exhaust gas发明人:Tsuru, Sumiaki,Yokoo, Akihiko, c/o AsahiKogaku Kogyo, k.k.,Sakurai, Takeshi, c/oYuugen Kaisha Fujimi Shoukai,Ogawa,Tetsuro, c/o Asahi Kogaku Kogyo, K.K.申请号:EP90107673.7申请日:19900423公开号:EP0393723A2公开日:19901024专利内容由知识产权出版社提供摘要:Deodorant comprising 5 to 100% by weight of a calcium phosphate compound having a molar ratio of Ca/P of 0.8 to 2.0, a deodorant sheet and filter sheet containing such calcium phosphate compound, and a functional paper containing 10 to 80% by weight of a calcium phosphate compound as well as a filtering medium for an exhaust gas which comprises a ceramic material containing 5 to 100% by weight of a calcium phosphate compound as well as a filter cartridge and exhaust gas filter containking the filtering medium. Based on excellent properties of the incorporated calcium phosphate compound, the agents and products according to the present invention exhibit a highly improved adsorptivity to an oil-soluble substance, odor substance, animal or vegetable cell, virus, biopolymeric substance or other substances and therefore can be advantageously used in various fields of technology. For example, the functional paper can be used as a deodorant, filter medium and adsorbent as well as flame-retardantpaper. The filtering medium, filter cartridge and filter can be used in an exhaust system to purity an exhaust gas produced therein.申请人:ASAHI KOGAKU KOGYO KABUSHIKI KAISHA,YUUGEN KAISHA FUJIMI SHOUKAI地址:36-9, Maeno-cho 2-Chome Itabashi-ku Tokyo 174 JP,23-35, Miyamotocho 2-Chome Tokorozawa-shi, Saitama-ken JP国籍:JP,JP代理机构:Schaumburg, Thoenes & Thurn更多信息请下载全文后查看。

MEMBERSHIP CARD SYSTEM ALLOWING MEDICAL EXAMINATIO

MEMBERSHIP CARD SYSTEM ALLOWING MEDICAL EXAMINATIO

专利名称:MEMBERSHIP CARD SYSTEM ALLOWINGMEDICAL EXAMINATION INFORMATION OFPET, CONTRAINDICATION MEDICINEINFORMATION, OR THE LIKE TO BEWRITTEN IN ELECTRONIC INFORMATIONSTORAGE AREA OF IC CHIP TYPEMEMBERSHIP CARD AND TO BE BROWSED,CONFIRMED AND CHANGED IN MEDICALINSTITUTION, AND ALLOWING PART OFPAID-OUT AMOUNT TO BE RETURNED发明人:NAKAMURA MORIHIDE,中村 守秀,OSHIMA JOJI,大島 譲二,AMAMIYA TORU,雨宮徹,KONISHI KEIKO,小西 啓子申请号:JP2008056969申请日:20080208公开号:JP2009187512A公开日:20090820专利内容由知识产权出版社提供专利附图:摘要:PROBLEM TO BE SOLVED: To provide a membership card allowing medical examination information of a pet to be browsed and referred to in any medical institution and usable to pay out as electronic money in each medical institution.SOLUTION: The medical examination information of the pet is written in a storage area in an IC chip body of the membership card containing a credit card type IC chip, and the medical examination information is read from a card body. The card includes an electronic money function, and when used to pay out in each medical institution, a card issuer is notified of an electronic money card management number managed by an electronic money operating entrepreneur, through the Internet or the like.COPYRIGHT: (C)2009,JPO&INPIT申请人:AMAMIYA TORU,雨宮 徹,NAKAMURA MORIHIDE地址:東京都中央区銀座6丁目14番5号 ギンザTS・サンケイビル5F 株式会社レノメディカ内国籍:JP更多信息请下载全文后查看。

BODY INSIDE OBSERVING DEVICE

BODY INSIDE OBSERVING DEVICE

专利名称:BODY INSIDE OBSERVING DEVICE发明人:KONO HIRONAO,河野 宏尚,UCHIYAMA AKIO,内山 昭夫,TAKIZAWA HIRONOBU,瀧澤 寛伸,KIKUCHI AKIRA,菊池 昭申请号:JP2003361782申请日:20031022公开号:JP2005124708A公开日:20050519专利内容由知识产权出版社提供专利附图:摘要:PROBLEM TO BE SOLVED: To observe an affected part in the digestive tract (especially in the intestine) without performing the intestinal lavage.SOLUTION: This body inside observation device 1 is provided with a capsuled casing 2orally input in the organism, an observation system 3 provided inside the casing 2 and observing the organism through an observation wall surface 2a of the casing 2, and a suction pump device 10 as a sticking assist means tightly sticking the observation wall surface 2a to the biotissue when observing.COPYRIGHT: (C)2005,JPO&NCIPI申请人:OLYMPUS CORP,オリンパス株式会社地址:東京都渋谷区幡ヶ谷2丁目43番2号国籍:JP代理人:棚井 澄雄,志賀 正武,青山 正和,鈴木 三義,高柴 忠夫更多信息请下载全文后查看。

Deodorants, deodorant sheets, filter sheets and fu

Deodorants, deodorant sheets, filter sheets and fu

专利名称:Deodorants, deodorant sheets, filter sheets and functional papers as well as filteringmediums for exhaust gas发明人:Tsuru, Sumiaki,Yokoo, Akihiko, c/o AsahiKogaku Kogyo, k.k.,Sakurai, Takeshi, c/oYuugen Kaisha Fujimi Shoukai,Ogawa,Tetsuro, c/o Asahi Kogaku Kogyo, K.K.申请号:EP90107673.7申请日:19900423公开号:EP0393723A2公开日:19901024专利内容由知识产权出版社提供摘要:Deodorant comprising 5 to 100% by weight of a calcium phosphate compound having a molar ratio of Ca/P of 0.8 to 2.0, a deodorant sheet and filter sheet containing such calcium phosphate compound, and a functional paper containing 10 to 80% by weight of a calcium phosphate compound as well as a filtering medium for an exhaust gas which comprises a ceramic material containing 5 to 100% by weight of a calcium phosphate compound as well as a filter cartridge and exhaust gas filter containking the filtering medium. Based on excellent properties of the incorporated calcium phosphate compound, the agents and products according to the present invention exhibit a highly improved adsorptivity to an oil-soluble substance, odor substance, animal or vegetable cell, virus, biopolymeric substance or other substances and therefore can be advantageously used in various fields of technology. For example, the functional paper can be used as a deodorant, filter medium and adsorbent as well as flame-retardantpaper. The filtering medium, filter cartridge and filter can be used in an exhaust system to purity an exhaust gas produced therein.申请人:ASAHI KOGAKU KOGYO KABUSHIKI KAISHA,YUUGEN KAISHA FUJIMI SHOUKAI地址:36-9, Maeno-cho 2-Chome Itabashi-ku Tokyo 174 JP,23-35, Miyamotocho 2-Chome Tokorozawa-shi, Saitama-ken JP国籍:JP,JP代理机构:Schaumburg, Thoenes & Thurn更多信息请下载全文后查看。

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TNF-␣–Induced Migration of Vascular Smooth Muscle CellsIs MAPK DependentStephan Goetze,Xiao-Ping Xi,Yasuko Kawano,Hiroaki Kawano,Eckart Fleck,Willa A.Hsueh,Ronald wAbstract—Migration of vascular smooth muscle cells(VSMC)is a key event in neointimal formation and atherosclerosis that may be linked to the accumulation of inflammatory cells and release of chemotactic cytokines.Tumor necrosis factor-␣(TNF-␣)induces chemotaxis of inflammatory cells and fibroblasts,but little is known about chemotactic signaling by TNF-␣in VSMC.The aim of this study was to investigate the role of TNF-␣in VSMC migration and to elucidate the chemotactic signaling pathways mediating this action.TNF-␣(50to400U/mL)induced migration of cultured rat aortic VSMC in a dose-dependent manner.Because activation of the extracellular signal-regulated kinase 1/2mitogen-activated protein kinase(MAPK)is known to be required in platelet-derived growth factor–directed and angiotensin II–directed migration of these cells,we used the MAPK-inhibitor PD98059to determine if chemotactic signaling by TNF-␣involves the MAPK pathway as well.We found that TNF-␣–directed migration was substantially inhibited by PD98059.TNF-␣(100U/mL)transiently activated MAPK with a maximal induction10minutes after stimulation that returned to baseline levels by2hours after treatment.Only a single peak of increased MAPK activity was seen.PD98059also blocked TNF-␣–stimulated MAPK activation in a concentration-dependent manner,which is consistent with its inhibition of TNF-␣–directed migration.To identify which TNF-␣receptor is involved in TNF-␣–induced MAPK activation,antibodies against the p55TNF-␣receptor-1(TNF-R1)and the p75TNF-␣receptor-2(TNF-R2)were used.VSMC express both receptors,but TNF-␣–induced MAPK activation was inhibited only by the TNF-R1antibody.The TNF-R2antibody had no effect.Thiazolidinediones are known to inhibit TNF-␣signaling in adipose tissue and attenuate platelet-derived growth factor–directed and angiotensin II–directed migration in VSMC.We therefore investigated the effects of the thiazolidinediones troglitazone(TRO)and rosiglitazone(RSG) on TNF-␣–induced migration.Both TRO and RSG inhibited migration,but neither attenuated TNF-␣–induced MAPK activation,indicating that their antimigration activity was exerted downstream of MAPK.These experiments provide the first evidence that early activation of MAPK is a crucial event in TNF-␣–mediated signal transduction leading to VSMC migration.Moreover,inhibition of TNF-␣–directed migration by the insulin sensitizers TRO and RSG underscores their potential as vasculoprotective agents.(Hypertension.1999;33[part II]:183-189.)Key Words:signal transductionⅢmuscle,smoothⅢatherosclerosisⅢMAPKⅢcytokineⅢmigrationV ascular smooth muscle cell(VSMC)migration is a critical event in lesion development and progression in restenosis and atherosclerosis.1Migration of VSMC is a complex response that occurs during several pathological processes that involve accumulation of inflammatory cells and the release of chemotactic cytokines.2Cytokines such as tumor necrosis factor-␣(TNF-␣)are pluripotent mediators of inflammation3and have been implicated in the chemotactic response of inflammatory cells4and fibroblasts.5Little is known,however,about the chemoattractant effects of TNF-␣in the vasculature.In addition to its role as an immune modulator,TNF-␣may also play an important role in atherogenesis and restenosis.6TNF-␣has been found to be expressed in VSMC after balloon injury7and in restenotic lesions8but not in the normal vasculature.Moreover,the presence of TNF-␣has been demonstrated in intimal VSMC9 and in plaques of atherosclerotic arteries10as well as in models of transplantation-associated atherosclerosis.11In an animal model of coronary-graft atherosclerosis,blockade of TNF-␣with a soluble TNF-␣receptor12inhibited acute coronary neointimal formation.Thus,cells of the arterial wall can both produce and respond to this cytokine in vivo.Thus TNF-␣is a highly important cytokine in the injured vascu-lature,where it may function to regulate the expression of growth factors(platelet-derived growth factor[PDGF],vas-cular endothelial cell growth factor[VEGF],fibroblastReceived September16,1998;first decision October14,1998;revision accepted October23,1998.From the Division of Endocrinology,Diabetes and Hypertension,University of California,Los Angeles,School of Medicine(S.G.,Y.K.,H.K., W.A.H.,R.L.);and the Department of Medicine/Cardiology,Virchow Klinikum,Humboldt University Berlin and German Heart Institute,Berlin, Germany(S.G.,E.F.).Correspondence to Ronald w,PhD,UCLA School of Medicine,Division of Endocrinology,Diabetes and Hypertension,Warren Hall,Second Floor,Suite24-130,900Veteran Ave,Box957073,Los Angeles,CA90095.E-mail rlaw@©1999American Heart Association,Inc.Hypertension is available at 183growth factor[FGF]),13,14adhesion molecules,15cytokines,16 and extracellular matrix degrading metalloproteinases17as well as directly affect VSMC growth and migration.To better define the role of TNF-␣in the injured vasculature,we investigated its effect on VSMC migration and identified the signaling pathways mediating this process.MethodsMaterialsDulbecco’s modified Eagle’s medium(DMEM),glutamine,antibi-otics,HEPES,DMSO,and monoclonal antibody against smooth muscle␣-actin were obtained from Sigma(St Louis,Mo);rat recombinant TNF-␣was from R&D systems(Minneapolis,Minn); and Hybond enhanced chemiluminescence nitrocellulose membrane, horseradish peroxidase–linked anti-rabbit antibody,and enhanced chemiluminescence Western blotting detection reagents were from Amersham Life Sciences(Arlington Heights,Ill).Fetal bovine serum (FBS)was purchased from Irvine Scientific(Santa Ana,Calif). Culture plastic ware and cell culture inserts(8␮m,24well-format; Falcon)and transwell chambers were obtained from Costar.Cell fixation and staining was performed with the Quik-Diff stain set from DADE.Sprague-Dawley rats were from Charles River,Mass.The M APK-E RK K inase(MEK)inhibitor PD98059and phos-phospecific and total-extracellular signal-regulated kinase(ERK)1/ ERK2mitogen-activated protein kinase(MAPK)rabbit antibodies were purchased from New England BioLabs(Beverly,Mass). Troglitazone(TRO)was kindly provided by Parke Davis;rosiglita-zone(RSG,formerly BRL49653)was a generous gift from Smith Kline Beecham.Antibodies against TNF-␣receptor-1and receptor-2 were obtained from Santa Cruz Biotechnologies(Santa Cruz,Calif). The bromodeoxyuridine(BrdU)dectection kit was purchased from Boehringer Mannheim.Cell CultureRat aortic smooth muscle cells from thoracic aortas of2-to 3-month-old Sprague-Dawley rats were prepared and cultured as described previously,18and the procedures followed were in accor-dance with institutional guidelines.A monoclonal antibody against smooth muscle␣-actin was used to assess the purity of the smooth muscle cell cultures.Flow cytometry of anti–smooth muscle␣-actin antibody–stained cells revealed a purity of95Ϯ3%.For all experi-ments early passaged(5or less)VSMCs were used,and each individual experiment represented in the n value was performed with an independent preparation of VSMC.MigrationVSMC migration was examined in transwell cell culture chambers with gelatin-coated polycarbonate membranes as described previous-ly.18In this assay,movement of VSMC through the coated mem-brane toward chemoattractants measures both invasion and chemo-taxis.Cells were pretreated with PD98059(10or30␮mol/L),TRO (1to20␮mol/L),RSG(0.1to10␮mol/L),or vehicle(0.4% FBS/DMEM)for30minutes at37°C.Cell attachment to the gelatin-coated membrane was not affected by any of the inhibitors (data not shown).Inhibitors were added to both the upper and the lower compartments and were present throughout the duration of the experiment.Migration was induced by addition of TNF-␣(50to400 U/mL)to the lower compartment.After a4-hour migration period, cells that had undergone migration toward the lower membrane surface were counted perϫ320high power field(HPF)with the use of an Axiovert135microscope.Four randomly chosen HPFs were counted per membrane.Experiments were performed in duplicate or triplicate and were repeated at least3times.Western Blot AnalysisFor analysis of phosphorylated and activated MAPK,cultured VSMC were grown to60%to70%confluence and then starved for 24hours in0.4%FBS/DMEM.For inhibitor studies,cells were pretreated for30minutes with PD98059(1to30␮mol/L),TNF-␣receptor-1antibody(1:100–1:1000),TNF-␣receptor-2antibody (1:100–1:1000),TRO(20␮mol/L),RSG(10␮mol/L),or vehicle (0.4%FBS/DMEM)alone,followed by the addition of TNF-␣(100 U/mL).After protein extraction,equal amounts of protein(30␮g) were separated by SDS-PAGE(7.5%standard gel),and Western blot analysis was performed as described previously18with rabbit poly-clonal antibodies that recognize either(a)ERK1or ERK2,which are phosphorylated on threonine202and tyrosine204,that is,phospho ERK/ERK2MAPK,or(b)all ERK1and ERK2proteins,indepen-dent of their phosphorylation state,that is,total ERK1/ERK2 MAPK,or goat polyclonal antibodies against TNF-␣receptor-1or TNF-␣receptor-2protein.Antibodies were used at a concentration of1:1000.To control for equal protein concentrations in MAPK experiments,2gels for each group were loaded in parallel with the same protein samples and blotted for activated,phosphorylated ERK1/ERK2MAPK or total ERK1/ERK2MAPK.All Western blot experiments were repeated at least twice with a different cell preparation.BrdU IncorporationTo determine the effect of TNF-␣on DNA-synthesis,incorporation of the thymidine analogue BrdU was measured19with the BrdU labeling and detection kit II from Boehringer Mannheim.VSMC were incubated either with TNF-␣(100U/mL)in serum-free medium or were kept in serum-free medium as control.Cell nuclei incorporating BrdU appeared brown and were counted in4to6 different HPFs per well and related to total cell number. StatisticsANOVA or paired or unpaired t test was performed for statistical analysis as appropriate.A probability valueϽ0.05was considered to be statistically significant.Data are expressed as meanϮSEM.ResultsTNF-␣Stimulates VSMC MigrationTo investigate the directed migration responses of VSMC toward TNF-␣,experiments were performed with a transwell migration chamber ing increasing concentrations of TNF-␣(50to400U/mL),we observed a dose-dependent increase in directed migration of cultured VSMC(Figure1). The cytokine induced a modest response at50U/mL with 7.2Ϯ1.2cells/HPF,representing a1.7Ϯ0.3-foldstimulation Figure1.VSMC migration toward increasing concentrations of TNF-␣(50to400U/mL).Migration of VSMC was determined with the use of a modified Boyden chamber assay as described in“Methods”and is shown in cell count per HPF.Results repre-sent at least3independent experiments done in duplicate or triplicate.Data are expressed as meanϮSEM.*PϽ0.05vs con-trol.Co indicates control.184TNF-␣–Directed Migration Is MAPK Dependentcompared with control (4.2Ϯ0.3cells/HPF).Significantly augmented migration occurred at 100U/mL and 200U/mL TNF-␣,leading to a 2.74Ϯ0.13-fold and 3.4Ϯ0.1-fold stim-ulation,respectively (both P Ͻ0.05compared with control).At the highest concentration tested,there was no further increase in migration toward TNF-␣(400U/mL)(3.38Ϯ0.14-fold over control,P Ͻ0.05).TNF-␣–Induced Migration Is MAPK-DependentAlthough an induction of the ERK1/ERK2MAPK pathway by TNF-␣in VSMC had not yet been demonstrated,MAPK activation is known to be an important step in PDGF-directed and angiotensin II–directed migration.18,20In addition,TNF-␣has been shown to induce MAPK activation in several other cell types including fibroblasts,21,22for which the cytokine is chemotactic.5We therefore tested the specific MEK inhibitor PD98059to examine a possible involvement of the MEK/MAPK pathway in TNF-␣–induced migration.The data in Figure 2demonstrate that treatment of VSMC with the MAPK pathway inhibitor PD98059at 10␮mol/L and 30␮mol/L significantly inhibited their response toward TNF-␣(100U/mL)(66Ϯ7.2%and 100Ϯ6%,respectively;P Ͻ0.05).At the concentrations used,PD98059did not cause any cytotoxic effects.There was no evidence of cell detach-ment or loss of plasma membrane integrity,as evidenced by the uptake of trypan blue.TNF-␣Transiently Activates MAPKTo corroborate our findings that the pharmacological inhibi-tor PD98059of the MAPK pathway inhibited TNF-␣–induced chemotactic signaling in VSMC,we examined the effect of TNF-␣on MAPK activation.VSMC were made quiescent by serum starvation,and after stimulation with TNF-␣the activation and phosphorylation of MAPK was assessed by immunoblotting with a phosphospecific ERK1/ERK2MAPK antibody.In parallel experiments,the amount of total ERK1/ERK2MAPK was determined in the same cellextracts with the use of an antibody that recognizes all ERK1/ERK2MAPKs independent of their phosphorylation state.Quiescent cells in the control groups exhibited low MAPK activity,as evidenced by the faint bands detected with the phosphospecific antibody (Figure 3).The residual MAPK activity in the controls probably is due to the fact that a small percentage of serum (0.4%)was present,which is known to stimulate MAPK.Stimulation with TNF-␣for 10minutes induced MAPK activation in a dose-dependent manner,resulting in a 11.5Ϯ2.3-fold increase in phosphorylated,activated MAPK compared with untreated control at a con-centration of 100U/mL (P Ͻ0.05).At higher concentrations of TNF-␣,no further increase in MAPK activation was observed,indicating that TNF-␣–inducible MAPK activity had plateaued (Figure 3).Interestingly,TNF-␣at 200U/mL induced more VSMC migration than was observed in cells stimulated with 100U/mL TNF-␣.Increased VSMC migra-tion at concentrations of TNF-␣higher than required to induce maximal MAPK activation therefore may be mediated through pathways other than MAPK.Since different growth factors and peptides activate MAPK either transiently (5to 10minutes peak)19or induce an additional second sustained peak in MAPK activity (5to 10minutes initial peak,followed by second peak 1to 2hours later),23,24we performed a time-course study of MAPK induction by TNF-␣.As shown in Figure 4,TNF-␣(100U/mL)induced a rapid and transient activation of MAPKthatFigure 2.Migration of VSMC toward TNF-␣is MAPK depen-dent.Cells were incubated with the MEK inhibitor PD98059for 30minutes before TNF-␣(100U/mL)was added.MAPK inhibi-tion with PD98059at 10␮mol/L significantly reduced migratory response and abolished TNF-␣–induced migration at 30␮mol/L.VSMC migration is shown as x-fold induction over control.Experiments were repeated 3times and done in duplicate.Data are expressed as mean ϮSEM.*P Ͻ0.05vs TNF-␣alone.Figure 3.TNF-␣stimulates MAPK activation in a dose-dependent manner.Quiescent VSMC were stimulated with increasing concentrations of TNF-␣(10to 400U/mL)for 10minutes.Equal amounts of protein (30␮g)were separated on 7.5%PAGE-SDS gels,followed by immunoblotting for (I)acti-vated,phosphorylated ERK1/ERK2MAPK or (II)total ERK1/ERK2MAPK (top,representative immunoblots).Results of den-sitometric analysis of 3different experiments are shown in the bottom panel.MAPK activity was measured by densitometry and is expressed in arbitrary units.Data are shown as mean ϮSEM.*P Ͻ0.05vs control.Co indicates control.Goetze et al January 1999Part II 185returns to baseline values within 60minutes.No second peak or sustained late phase in TNF-␣–stimulated MAPK activity was detected.TNF-␣did not affect the amount of total MAPK protein during the investigated time course (Figure 4lane II).Treatment with PD98059(1to 30␮mol/L)blocked TNF-␣–stimulated MAPK activation in a concentration-dependent manner (Figure 5).TNF-␣–induced MAPK activation was inhibited by 37Ϯ5%and 74Ϯ8%at 1␮mol/L and 10␮mol/L PD98059,respectively.At 30␮mol/L,PD98059completely abolished the effect of TNF-␣on MAPK activation (P Ͻ0.05).TNF-␣Stimulates MAPK in VSMC Through p55TNF-␣Receptor-1To identify the receptor that mediates TNF-␣–induced MAPK activation,quiescent cells were pretreated with dif-ferent concentrations of antibodies against the p55TNF-␣receptor-1and the p75TNF-␣receptor-2and then stimulated with TNF-␣(100U/mL,10minutes).Whereas TNF-␣–stimulated MAPK activity was inhibited by increasing con-centrations of the TNF-␣receptor-1antibody (38Ϯ7%inhi-bition at 1:500and 68Ϯ11%inhibition at 1:100),the TNF-␣receptor-2antibody had no effect (Figure 6).An additional Western blot analysis of the same control extracts confirmed the presence of both receptors in VSMC (Figure 7).Thus,TNF-␣specifically activates MAPK in VSMC through the p55TNF-␣receptor-1.Thiazolidinediones Inhibit VSMC Migration Toward TNF-␣Thiazolidinediones (TZDs)such as TRO are antidiabetic insulin sensitizers that have been reported to inhibit TNF-␣signaling in adipose tissue.25In addition,we have previously shown that TRO inhibits PDGF-mediated andangiotensinFigure 4.MAPK activation by TNF-␣is monophasic and tran-sient.Serum-starved VSMC were treated with TNF-␣(100U/mL)for 10to 180minutes,and protein samples were immunoblotted with I,a phosphospecific ERK1/ERK2MAPK antibody,or II,an antibody against total ERK1/ERK2MAPK.Western blots shown are representative of 4experiments with different cell prepara-tions.Co indicatescontrol.Figure 5.PD98059dose-dependently inhibits TNF-␣–induced MAPK activation.Cells were incubated with MEK-inhibitor PD98059(1to 30␮mol/L)for 30minutes before TNF-␣(100U/mL)was added.After stimulation with TNF-␣for 10minutes,cell lysates were immunoblotted for I,activated,phosphorylated ERK1/ERK2MAPK,or II,total ERK1/ERK2MAPK (top,repre-sentative immunoblots).Results of densitometric analysis of 3different experiments are shown in the bottom panel.MAPKactivity was measured by densitometry and is expressed in arbi-trary units.Data are shown as mean ϮSEM.*P Ͻ0.05vs TNF-␣alone.Co indicatescontrol.Figure 6.TNF-␣activates MAPK in VSMC through the p55TNF-␣receptor-1.Quiescent rat aortic VSMC were pretreated for 30minutes with increasing concentrations of p55TNF-␣receptor-1antibody (1:1000–1:100)or p75TNF-␣receptor-2antibody (1:1000–1:100)followed by addition of TNF-␣(100U/mL).After coincubation with TNF-␣and receptor antibodies for 10minutes,cells were harvested and protein samples were immunoblotted with I,a phosphospecific ERK1/ERK2MAPK antibody,or II,an antibody against total ERK1/ERK2MAPK.Western blots shown are representatives of 4experiments with different cell preparations.Co indicatescontrol.Figure 7.Both TNF-␣receptors are present in VSMC.Control cell extracts were separated on 7.5%PAGE-SDS gels,followed by Western blot analysis with antibodies against p55TNF-␣receptor-1or p75TNF-␣receptor-2.As shown,distinct bands of the right sizes were detected for both TNF-␣receptors.Experiment was repeated with a different set of protein samples,revealing the same results.186TNF-␣–Directed Migration Is MAPK DependentII–mediated migration of VSMC.19,20Because of those find-ings,we examined the effects of the TZDs TRO and rosigli-tazone RSG on TNF-␣–induced migration.The data in Figure 8show that migration of VSMC toward the cytokine was significantly inhibited by treatment with TRO 10␮mol/L and 20␮mol/L by 68Ϯ5.8%and 99Ϯ7.2%,respectively (both P Ͻ0.05vs TNF-␣100U/mL alone).An even more potent effect was observed for RSG that completely abolished migration toward TNF-␣at 10␮mol/L (P Ͻ0.05)(Figure 8).At all concentrations of TRO (1to 20␮mol/L)or RSG (0.1to 10␮mol/L)used in migration assays,we observed no cytotoxic effects evidenced by the lack of cell detachment or the absence of a significant number of cells staining posi-tively for trypan blue.TZDs Act Downstream of TNF-␣–Induced MAPK ActivationTo determine whether TRO and RSG inhibited migration by targeting the MAPK pathway,we investigated their effect on TNF-␣–induced MAPK activation.VSMC were made quies-cent by 24-hour serum starvation,then pretreated with TRO or RSG for 30minutes,followed by stimulation with TNF-␣(100U/mL)for 10minutes.Neither TRO nor RSG attenuated the MAPK activation in response to TNF-␣,indicating that the TZDs inhibit VSMC migration downstream of MAPK activation,a signaling step required for TNF-␣–directed migration (Figure 9).TNF-␣Is Not Mitogenic for VSMCBecause VSMC accumulation in restenosis and atherosclero-sis results from a combination of cell growth and migration,it was of interest to determine whether TNF-␣also functions as a mitogen for ing the thymidine analogue BrdU,we examined the effect of TNF-␣on VSMC proliferation.Treatment with TNF-␣(100U/mL)for 24hours did notresult in a significant increase in DNA-synthesis compared with control,as determined by BrdU-incorporation (TNF-␣:9.8Ϯ0.9%;control:8.4Ϯ0.7%).DiscussionTNF-␣is a cytokine that is released by inflammatory cells at sites of vascular injury and is expressed in the arterial wall under pathological conditions,in which it is associated with lesion formation.3,6–11Migration of VSMC from the tunica media to the intima is one of the major pathological vascular responses that leads to the development and progression of intimal thickening.1Previous studies demonstrated that TNF-␣is a potent migration factor for different cell types,such as fibroblasts and inflammatory cells.4,5The present investigation demonstrates that (1)TNF-␣is a potent migration factor but not a growth factor for VSMC;(2)TNF-␣stimulates only an early activation (10minutes)of MAPK but not a second,delayed peak in activity (2to 6hours),which may explain why this cytokine is a potent migration factor but a weak mitogen;(3)TNF-␣–induced MAPK activation is mediated by the p55TNF-␣receptor and not the p75TNF-␣receptor;and (4)TZDs inhibit TNF-␣–stimulated VSMC migration.The mechanisms and intracellular signaling pathways lead-ing to VSMC migration are not completely understood.Among the cytosolic events in response to migration factors,recent studies have recognized the ERK1/ERK2MAPKs as key signaling steps for this process in VSMC.18,19,26How-ever,migration of VSMC has also been linked to other signaling molecules,such as increased phosphatidylinositol turnover leading to the activation of phospholipase C 27as well as increased intracellular calcium and activation of the calcium/calmodulin-dependent kinase II.28–31It has been shown that PDGF-induced chemotactic signaling involves activation of the calcium/calmodulin kinase II,which in turn leads to phosphorylation and activation of myosin light chain kinase.32Yet,another recent study revealed that myosin light chain kinase,which phosphorylates and reorganizes cytoskel-etal components that facilitate cell movement,33is also a substrate for MAPK.34,35The importance of the MAPK-pathway in VSMC migration is supported by findings show-ing that inhibition of MAPK with antisenseoligodeoxynucle-Figure 8.Migration of VSMC toward TNF-␣is inhibited byTZDs troglitazone (TRO,1to 20␮mol/L)and rosiglitazone (RSG,0.1to 10␮mol/L)in a concentration-dependent manner.Migra-tion responses are shown as x-fold induction over control.Results represent 4independent experiments done in duplicate.Data are expressed as mean ϮSEM.*P Ͻ0.05vs TNF-␣alone.Figure 9.TZDs do not affect TNF-␣–induced MAPK activation.After a 30-minute preincubation with TRO (20␮mol/L)or RSG)10␮mol/L),quiescent cells were stimulated with TNF-␣(100U/mL)for 10minutes.Immunoblotting was performed as described in Figure 3.I,Phosphospecific ERK1/ERK2MAPK antibody;II,antibody against total ERK1/ERK2MAPK.Western blots shown are representative of 2independently performed experiments.Goetze et al January 1999Part II 187otides or the MEK-inhibitor PD98059blocks PDGF-directed and angiotensin II–directed migration.18,36TNF-␣is known to activate the MAPK pathway in fibroblasts22and inflammatory cells21;however,nothing was known about its effects on MAPK in ing the pharmacological inhibitor PD98059,we were able to identify that the activation of ERK1/ERK2MAPK is a critical signaling step for TNF-␣–directed migration of VSMC. PD98059selectively inhibits the dual-specificity kinase MEK,which phosphorylates and activates MAPK.In our experiments,PD98059,at the highest concentration tested, completely abolished both TNF-␣–induced MAPK activity and VSMC migration.The MEK inhibitor appears to be highly selective in blocking only the MAPK pathway;several studies have shown that it does not affect a number of other signaling proteins.37–39A variety of growth factors and peptides activate MAPK in different ways,leading either to a transient activation within 5to10minutes after stimulation19or to a biphasic response with a sustained second peak appearing1or2hours later.23,24 A recent study investigating the functional relevance of these kinetics reports that the early peak in PDGF-induced MAPK activation is crucial for VSMC migration,whereas the sus-tained second phase of MAPK activity is important for mitogenesis.26Potent chemoattractants such as PDGF18and thrombin40that activate MAPK in a biphasic manner23,24are also mitogenic for VSMC.41,42Our data show that TNF-␣induces only the early peak of MAPK activity,leading to directed migration.No second peak of MAPK activity was observed,and consistent with other studies,43we saw no effect of TNF-␣to stimulate VSMC proliferation.However, some growth factors,such as FGF,which rapidly activate MAPK,do not induce migration of VSMC.19This may indicate that MAPK activation is necessary but not sufficient for migration.To further elucidate the signaling steps involved with TNF-␣–induced MAPK activation,we examined the role of the TNF-␣receptors.In other cell types,2main TNF-␣receptors have been described:the p55TNF-␣receptor-1 (TNF-␣-R1),and the p75TNF-␣receptor-2(TNF-␣-R2).44 Although many cell types express both receptors,45the majority of TNF-␣–induced signaling events are mediated by TNF-␣-R1.46Using antibodies to neutralize the TNF-␣-R1 and the TNF-␣-R2,we found that only the TNF-␣-R1 antibody blocked MAPK activation by TNF-␣,whereas the TNF-␣-R2had no effect.This finding is consistent with another study that showed that TNF-␣-R1mediated MAPK activation in HeLa cells.45In VSMC,TNF-␣induces signal-ing through the TNF-␣-R1that leads to MAPK activation, which is required for cell migration.In addition to its role in directed migration toward TNF-␣and other chemoattractants,MAPK is also involved in mito-genic signaling by growth factors in VSMC.VSMC prolif-eration and migration both importantly contribute to the accumulation of these cells in vascular lesions.A potential candidate for a therapeutic approach for preventing lesion formation in the arterial wall could be the novel insulin-sensitizing class of TZDs.In the present study,we were able to demonstrate that2TZDs,TRO and RSG,inhibited TNF-␣–directed migration of VSMC.Furthermore,we pre-viously showed that TRO inhibits PDGF-directed and angio-tensin II–directed migration in VSMC19,20and that migratory responses toward both chemoattractants are MAPK depen-dent.18,36The precise mechanism by which TZDs inhibit MAPK-dependent migration pathways remains to be eluci-dated,although our results suggest an effect downstream of MAPK,since neither TRO nor RSG affected TNF-␣–stimu-lated MAPK activation.Because of the enormous clinical relevance of VSMC accumulation in the development and progression of athero-sclerosis and restenosis,strategies targeting the MAPK path-way as a common signaling step in VSMC migration and proliferation may provide new therapeutic approaches for the treatment and prevention of these vascular diseases.AcknowledgmentsThis study was supported by the Deutsche Forschungsgesellschaft (DFG GO800/1-1)and the NIH(HL-58328-03).The authors would like to thank Verena Fromm for assistance in preparing the manuscript.References1.Schwartz SM.Perspectives series:cell adhesion in vascular biology:smooth muscle migration in atherosclerosis and restenosis.J Clin Invest.1997;99:2814–2816.2.Abedi H,Zachary I.Signalling mechanisms in the regulation of 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acute coronary artery neointimal formation.Circulation.1994;89:2768–2779.13.Winkles JA,Gay CG.Regulated expression of PDGF A-chain mRNA inhuman saphenous vein smooth muscle cells.Biochem Biophys Res Commun.1991;180:519–524.14.Yoshida S,Ono M,Shono T,Izumi H,Ishibashi T,Suzuki H,Kuwano M.Involvement of interleukin-8,vascular endothelial growth factor,and basic fibroblast growth factor in tumor necrosis factor alpha-dependent angiogenesis.Mol Cell Biol.1997;17:4015–4023.188TNF-␣–Directed Migration Is MAPK Dependent。

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