Urokinase-type plasminogen activator promotes synaptic repair in the ischemic brain

The central nervous system has a very high energy requirement. Accord- ingly, despite representing only 2% of the body＇s mass, the brain uses 20% of the total oxygen consumption. Importantly, because most of this energy is used to maintain synaptic activity, even a mild decrease in its supply to the brain has deleterious implications for synaptic function.

Urokinase-type plasminogen activator receptor as a predictor of poor outcome in patients with systemic inflammatory response syndrome

BACKGROUND:Urokinase-type plasminogen activator(uPA) and urokinase-type plasminogen activator receptor(uPAR) are known as important factors,which mediate a variety of functions in terms of vascular homeostasis,inflammation and tissue repair.However,their role in systemic inflammatory response syndrome(SIRS) has been less well studied.This study aimed to test the hypothesis that the abnormalities of fibrinolysis and degradation of extracellular matrix mediated by uPA and uPAR are directly related to the patients with SIRS.We therefore analyzed their role and clinicopathological significance in patients with SIRS.METHODS:A case-control study was conducted with 85 patients who were divided into two groups according to the diagnostic criteria of SIRS:SIRS group(n=50) and non-SIRS group(/7=35).The SIRS group was divided into MODS group(n=26) and non-MODS group(n=24) by their severity,and survival group(n=35) and non-survival group(n=15) by their prognosis.Another 30 healthy adults served as normal controls.uPA and uPAR in plasma were detected by commercial enzyme-linked immunosorbent assay(ELISA) kits.RESULTS:The plasma level of uPA was lower in the SIRS group than in the non-SIRS group and controls(P<0.001 and P<0.001).It was lower in sepsis patients and the MODS group than in the non-sepsis patients and the non-MODS patients(all P<0.05).However,there was no difference in uPA level between survivors and non-survivors(P>0.05).The plasma level of uPAR increased in the SIRS group compared with the non-SIRS group and controls(P<0.001 and P<0.001).There was a significant elevation of uPAR in sepsis patients,MODS patients and non-survivors as compared with non-sepsis patients,non-MODS patients and survivors respectively(all P<0.05).Plasma uPAR levels were positively correlated with APACHE Ⅱ score(r=0.575,P<0.001) and SOFA score(r=0.349,P=0.013).AUCs for the prediction of SIRS mortality were 0.67 and 0.51,respectively,for uPA and uPAR.CONCLUSION:uPAR could be a predictor of poor outcome in patients with SIRS.

Crystal Structures of 2-Aminobenzothiazole-based Inhibitors in Complexes with Urokinase-type Plasminogen Activator

Urokinase-type plasminogen activator （uPA） plays a crucial role in the regulation of plasminogen activation, tumor cell adhesion and migration. The inhibition of uPA activity is a promising mechanism for anti-cancer therapy. Most current uPA inhibitors employ a highly basic group （either amidine or guanidine group） to target the S1 pocket of uPA active site, which leads to poor oral bioavailability. Here we study the possibility of using less basic 2-aminobenzothiazole （ABT） as S1 pocket binding group. We report the crystal structures of uPA complexes with ABT or 2-amino-benzothiazole-6-carboxylic acid ethyl ester （ABTCE）. The inhibitory constants of these two inhibitors were measured by a chromogenic competitive assay, and it was found that ABTCE is a better inhibitor for uPA （Ki = 656 μM） than ABT （Ki = 5.03 mM）. This work shows that 2-amniobenzothiazole can be used as P1 group which may have better oral bioavailability than the commonly used amidine or guanidine group. We also found the ethyl ester group occupies the characteristic oxyanion hole and contacts to uPA 37- and 60-loops. Such work provides structural information for further improvements of potency and selectivity of this new class of uPA inhibitor.

Role of Urokinase-type Plasminogen Activator in the Precontact Sperm-egg Communication and Fertility of Mice in vitro

Objective To explore the role of urokinase-type plasminogen activator（uPA） in precontact sperm-egg communication and fertility of mice in vitro. Methods Firstly, sperm chemotaxis （SC） induced by uPA was assayed by measuring the sperm densities in capillaries with a descending gradient or no gradient of uPA respectively. Secondly, the role of uPAR that exists in sperm plasma membrane in SC was studied by examining the change of sperm density in capillary after incubating spermatozoa with anti-uPAR antibody. Thirdly, SC induced by eggs, which had been treated with uPA, PAl-1 and anti-uPAR beforehand respectively, was assayed to study the role of uPA in PSEC. Lastly, the fertilization capability of spermatozoa treated with uPA was examined by counting the number of fertilized eggs. Results 1）The density of spermatozoa that migrated down the gradient of uPA into the capillary was significantly lower than that into the capillary containing no-gradient uPA. 2） When uPAR of spermatozoa was inhibited by anti-uPAR antibody, the density of spermatozoa that migrated into the capillary with ascending gradient of uPA decreased correspondingly. 3） The density of spermatozoa attracted by eggs, which were treated with uPA beforehand, increased significantly than that of attracted by non-treated eggs. On the contrary, the sperm density decreased correspondingly when the egg was treated with PAI-1. 4） The number of fertilized eggs increased significantly after the spermatozoa used here was treated with uPA beforehand. Conclusion uPA could induce SC of mice sperm in vitro through the uPAR on its membrane, enhance the capability of egg inducing SC, and promote spermatozoa to fertilize eggs. Thus, uPA may act as an attractant in PSEC, increase the chance encounter of spermatozoa and eggs, therefore, enhance the fertility success correspondingly. This study, in some degree, provides an evidence that uPA may be used as a new medicine and diagnostic reagent for male infertility.

EXPRESSION AND SIGNIFICANCE OF UROKINASE-TYPEPLASMINOGEN ACTIVATOR IN BREAST CANCER

Objective: To study the expression and clinical significance of urokinase-type plasminogen activator (uPA) in breast cancer. Methods: Applying streptavidin-biotin complex (SABC) immunohistochemical technique, expression of uPA was studied in 100 patients with primary breast cancer. Results: There were 55 patients with high uPA expression, and 45 with lower expression. There was significant correlation between uPA expression and TNM stage, lymph node status, and the tumor size. Neither age, menopausal status, nor ER status was significantly related with level of uPA expression. The patients with high expression of uPA had significantly shorter disease-free survival (DFS) and overall survival (OS) than did those with low expression of uPA. Univariate analysis showed that uPA as a prognostic factor was of similar magnitude to lymph node status and TNM stage, but stronger than that of ER status and tumor size. UPA was an independent prognostic factor affecting disease-free survival and overall survival. Conclusion: uPA appears to be a strong and independent biologic marker for predicting prognosis of breast cancer.

Crystal Structures of Urokinase-type Plasminogen Activator in Complex with 4-(Aminomethyl) Benzoic Acid and 4-(Aminomethyl-phenyl)-methanol

Urokinase-type plasminogen activator （uPA） is a trypsin-like serine protease and plays a key role in several biological processes, including tissue remodeling, cell migration, and matrix degradation. The inhibitors of uPA have been shown to prevent the spread of metastasis and tumor growth, and accordingly uPA is widely recognized as a target for the treatment of cancer. In this work, we report the crystal structures of the complexes of uPA with its inhibitors： 4- （aminomethyl）-benzoic acid （AMBA） and 4-（aminomethyl-phenyl）-methanol （AMPM）, both at a resolution of 2.35 А. The inhibitory constants of these two inhibitors were measured by a chromogenic competitive assay, and it was found that AMBA is a better inhibitor for uPA （Ki = 2.68 mM） than AMPM （Ki = 13.99 mM）. The structural study shows that the binding mode of inhibitor AMBA on uPA is similar to that of AMPM on uPA, both docked into the active site S1 pocket of uPA. Structural details of these complexes are provided to explain the difference of inhibitory constants.

The plasminogen activating system in the pathogenesis of Alzheimer’s disease

Dementia is a clinical syndrome that affects approximately 47 million people worldwide and is characterized by progressive and irreversible decline of cognitive,behavioral and sesorimotor functions.Alzheimer’s disease(AD)accounts for approximately 60–80%of all cases of dementia,and neuropathologically is characterized by extracellular deposits of insoluble amyloid-β(Aβ)and intracellular aggregates of hyperphosphorylated tau.Significantly,although for a long time it was believed that the extracellular accumulation of Aβwas the culprit of the symptoms observed in these patients,more recent studies have shown that cognitive decline in people suffering this disease is associated with soluble Aβ-induced synaptic dysfunction instead of the formation of insoluble Aβ-containing extracellular plaques.These observations are translationally relevant because soluble Aβ-induced synaptic dysfunction is an early event in AD that precedes neuronal death,and thus is amenable to therapeutic interventions to prevent cognitive decline before the progression to irreversible brain damage.The plasminogen activating(PA)system is an enzymatic cascade that triggers the degradation of fibrin by catalyzing the conversion of plasminogen into plasmin via two serine proteinases:tissue-type plasminogen activator(tPA)and urokinase-type plasminogen activator(uPA).Experimental evidence reported over the last three decades has shown that tPA and uPA play a role in the pathogenesis of AD.However,these studies have focused on the ability of these plasminogen activators to trigger plasmin-induced cleavage of insoluble Aβ-containing extracellular plaques.In contrast,recent evidence indicates that activity-dependent release of uPA from the presynaptic terminal of cerebral cortical neurons protects the synapse from the deleterious effects of soluble Aβvia a mechanism that does not require plasmin generation or the cleavage of Aβfibrils.Below we discuss the role of the PA system in the pathogenesis of AD and the translational relevance of data published to this date.

Chemotactic effect of urokinase-type plasminogen activator on mouse spermatozoa in vitro

The aim of this study is to investigate the che-motactic effect of urokinase-type plasminogen activator(uPA)on mouse spermatozoa.Capillary assays were applied to study the chemotactic activity of ascending and descending gradients of uPA.Firstly,the chemotactic effect of an ascending gradient of uPA on mouse sper-matozoa was observed by counting the number of sper-matozoa that migrated into the capillary after incubation with uPA for 5,10,20,and 30 min,respectively,com-pared with that after incubation with F10.Twenty min-utes was a suitable incubation time to obtain a plateau of sperm accumulation.Meanwhile,to confirm the specific effect of uPA on mouse sperm chemotaxis,uPA inhibitor(PAI-1)and anti-uPAR rabbit IgG were added to the test solution containing 20 U/mL uPA,respectively.To exclude the possibility that PAI-1 and anti-uPAR rabbit IgG may affect sperm accumulation nonspecifically,PAI-1 and anti-uPAR rabbit IgG were added to F10,respect-ively.It was found that the chemotactic effect of uPA was neutralized completely by PAI-1 and anti-uPAR rabbit IgG.PAI-1 and anti-uPAR rabbit IgG had no neutral-izing effect on the sperm chemotactic effect.Lastly,the sperm chemotaxis response to a descending gradient of uPA was also observed.Taken together,the results sug-gest that uPA can induce sperm chemotaxis in vitro by binding to its receptor on the sperm membrane and may act as a chemoattractant in precontacting sperm-egg com-munication thereby increasing the chance encounter of spermatozoa and eggs.

尿激酶型纤溶酶原激活剂(uPA)和其受体(uPAR)在肝细胞癌的表达及其临床和病理意义

目的 研究尿激酶型纤溶酶原激活剂 (uPA)和其特异性受体 (uPAR)在肝细胞癌 (HCC)的表达及其临床和病理意义。 方法 应用免疫组织化学和酶联免疫吸附法 (ELISA)检测uPA和uPAR在HCC和非癌组织中的表达 ,并与临床及病理指标相联系。 结果 HCC中uPA和uPAR的阳性表达率分别为 75 0 % (12 / 16 )和 6 8.8% (1/ 16 )。在进展性和侵袭性肿瘤与非进展性和非侵袭性肿瘤间uPA和uPAR阳性率无显著性差异 (P >0 .0 5 )。阳性染色主要位于癌细胞和基质细胞胞浆且主要位于癌侵犯的边缘。非癌组织仅见少量阳性染色细胞 ,对照组无特异性阳性染色。在ELISA中 ,癌与非癌组织uPA抗原水平分别为 (4 2 3± 0 5 7)ng/mg蛋白和 (1 2 6± 0 14)ng/mg蛋白 ,uPAR抗原水平分别为 (4 2 5± 0 2 1)ng/mg蛋白和 (3 15± 0 2 3)ng/mg蛋白 ,有显著性差异 (t=18.96 3,P =0 .0 0 0 ;t=13.6 93 ,P =0 .0 0 0 )。且进展性和侵袭性肿瘤中其抗原水平显著高于非进展性和非侵袭性肿瘤 (P <0 0 5 )。 结论 uPA和uPAR在肝细胞癌表达较高并与其进展和侵袭密切关联。

上皮性卵巢癌组织中uPA和PAI-1的表达及意义

背景与目的:近年研究表明,尿激酶型纤溶酶原激活物(urokinase-typeplasminogen activator,uPA)及其抑制物(plasminogen activator inhibitor,PAI)在肿瘤侵袭、转移过程中起重要作用,但其与上皮性卵巢癌的关系研究甚少。本研究从蛋白水平探讨uPA、PAI-1在上皮性卵巢癌浸润、转移中的作用,组织中的分布情况、及与预后的关系。方法:应用免疫组化法检测80例上皮性卵巢癌、20例良性卵巢肿瘤组织uPA、PAI-1蛋白表达,并结合临床病理因素、预后进行分析。结果:上皮性卵巢癌、良性卵巢肿瘤组织中uPA阳性率分别为77.5%和30.0%(P<0.001),PAI-1阳性率分别为55.0%和20.0%(P=0.005)。上皮性卵巢癌组织中uPA表达与PAI-1表达呈显著性正相关(P=0.001)。uPA阳性与盆腹腔转移病灶直径>1cm有关(P=0.038),与患者年龄、FIGO分期、组织学类型、病理分级、术前血CA125值、卵巢病灶大小、残留病灶大小无显著性相关(P>0.05);PAI-1阳性与FIGO分期有显著性相关(P=0.022),与上述其他临床病理因素无显著性相关(P>0.05)。多因素Cox回归模型显示,uPA表达是肿瘤无进展生存、总生存的独立危险因素;PAI-1表达是总生存的独立危险因素。结论:上皮性卵巢癌组织中uPA、PAI-1表达上调。uPA、PAI-1有可能作为预测上皮性卵巢癌预后的参考指标。

uPA、uPAR、PAI-1血浆含量与卵巢恶性肿瘤的相关性研究

目的探讨尿激酶型纤溶酶原激活物(uPA)及其受体(uPAR)和抑制剂(PAI-1)血浆含量与卵巢恶性肿瘤之间的关系。方法收集52例卵巢恶性肿瘤患者血液标本,以30例健康人作对照,用ELISA法分别检测uPA、uPAR和PAI-1的含量。结果uPA、uPAR在卵巢恶性肿瘤各期之间均有极显著性差异(P<0.01),PAI-1在卵巢恶性肿瘤FIGOⅠ～Ⅲ期的含量逐渐升高,但在FIGOⅣ期时显著下降(P<0.05)。患者组uPA、uPAR、PAI-1均较对照组升高,差异极显著(P<0.01)。结论uPA、uPAR在卵巢恶性肿瘤患者可作为预后的判断指标,PAI-1与卵巢恶性肿瘤的分期有一定的相关性。

乏氧对人舌癌Tca8113细胞uPA活性的影响

目的:研究乏氧对人舌癌Tca8113细胞尿激酶型纤溶酶原激活物(uPA)活性的影响。方法:根据人舌癌Tca8113细胞不同乏氧时间,分为0、6、12、24h4组,分别给予乏氧。采用免疫组化检测Tca8113细胞uPA蛋白表达,原位杂交方法检测Tca8113细胞uPA mRNA表达,并用Spot及IPP图像采集分析系统处理原位杂交染色结果。结果:Tca8113细胞uPA阳性表达呈棕黄色,定位于细胞质和细胞膜。Tca8113细胞uPAmRNA阳性表达呈紫蓝色,定位于细胞质;随着乏氧时间逐渐延长,紫蓝色着色逐渐加深变浓;吸光度逐渐增大,且两两比较均有显著性差异(P<0.05)。结论:乏氧可促进人舌癌Tca8113细胞uPA mRNA高表达。提示:乏氧可能通过激活肿瘤细胞高表达uPA而促进口腔癌侵袭和转移。

uPA和MMP-9在乳腺浸润性导管癌中的表达及其相关性

目的探讨尿激酶型纤溶酶原激活剂(uPA)和基质金属蛋白酶(MMP-9)在乳腺癌及肿瘤周围组织中的表达与乳腺癌侵袭、转移的关系。方法应用免疫组化Envision法检测50例乳腺癌及20例癌旁组织中uPA和MMP-9蛋白的表达情况,并分析其相关性及与临床病理特征进行比较。结果乳腺癌组织中uPA的阳性表达率为60%(30/50),MMP-9的阳性表达率为66%(33/50),显著高于癌旁组织(P<0.01),在乳腺癌组织中uPA、MMP-9蛋白表达之间存在显著正相关(r=0.630,P<0.01)。uPA、MMP-9表达与淋巴结转移、临床分期显著相关(P<0.05),与患者的年龄、肿瘤大小无明显相关性(P<0.05)。结论乳腺癌组织中存在uPA、MMP-9的高表达;uPA蛋白可通过诱导MMP-9蛋白的表达上调,促进乳腺癌侵袭、转移。

uPA、Cath-D蛋白在胃癌中的表达及临床意义

目的探讨胃癌组织中尿激酶型纤维蛋白溶解酶原激活剂(uPA)和组织蛋白酶D(Cath-D)在胃癌中的表达及其与胃癌临床病理特征的关系。方法运用免疫组化SP法检测68例胃癌组织及31例正常胃黏膜组织中uPA和Cath-D的表达。结果胃癌组织中uPA和Cath-D的表达率分别为69.1%和77.9%,高于正常胃黏膜中uPA和Cath-D的表达率(分别为25.8%和32.3%),差异均有统计学意义(P<0.01);uPA阳性表达与胃癌的浸润深度、淋巴结转移及临床分期有关;Cath-D阳性表达与胃癌的分化程度、浸润深度、淋巴结转移及临床分期有关;uPA表达与Cath-D表达呈正相关。结论 uPA和Cath-D的表达与胃癌的浸润及转移密切相关,uPA和CATH-D的表达具有协同性。两者的联合检测有助于判断胃癌发展及预后。

uPA在鼻咽癌组织中的表达及其意义

目的探讨尿激酶型纤溶酶原激活剂在鼻咽癌组织中的表达及其与鼻咽癌侵袭和转移的关系。方法应用RT—PCR和免疫组化方法检测55例鼻咽癌术后组织标本中uPA的表达，分析uPA表达与鼻咽癌临床病理特征的关系。结果鼻咽癌组织中都存在uPA基因的表达，并定位于细胞质和／或细胞膜中，uPA基因mRNA和蛋白质的检测均显示有淋巴结转移的癌组织uPA表达高于无淋巴结转移组，TNM分期Ⅲ～Ⅳa的癌组织高于Ⅰ～Ⅱ组，均有统计学意义。结论鼻咽癌组织中uPA基因及蛋白质表达与鼻咽癌的侵袭和转移密切相关。

uPA和V-ATP酶mRNA在肝癌组织中的表达及意义

目的探讨uPA、V-ATP酶mRNA在肝细胞癌中的表达及其意义。方法用RT-PCR方法检测45例肝细胞癌及其癌旁组织uPA和V-ATP酶mRNA表达,以7例肝血管瘤作为对照组。结果uPA mRNA在癌组、癌旁组及对照组的阳性率分别为100%、84.4%和28.6%,3组比较差异显著(P<0.05);V-ATP酶mRNA在癌组、癌旁组及对照组的阳性率分别为100%、86.7%、57.1%,3组比较均差异显著(P<0.05);以有/无肝内静脉癌栓形成和肝外转移将癌组分为转移和无转移两组,uPA、V-ATP酶mRNA在两组的表达均有差异显著(P<0.01);而它们的表达与乙型肝炎、病理分级、血清AFP水平以及肿瘤直径无明显关系(P>0.05)。结论肝细胞癌中uPA、V-ATP酶mRNA高表达与肝癌的侵袭转移有关,有可能成为临床治疗的靶点。

甘糖酯对大鼠尿激酶型纤溶酶原激活物(uPA)mRNA水平的影响

从分子水平上探讨海洋新药甘糖酯的抗栓作用机理。实验以大鼠为材料，采用定量ＲＴＰＣＲ方法定量检测口服甘糖酯后大鼠尿激酶型纤溶酶原激活物（ｕＰＡ）基因ｍ ＲＮＡ水平变化。研究表明大鼠口服甘糖酯后ｕＰＡ基因ｍ ＲＮＡ 水平高于对照组，差异显著。结论认为甘糖酯可提高体内ｕＰＡ基因的ｍ ＲＮＡ 水平，从而提高ｕＰＡ蛋白的活性，激活机体的纤溶系统，达到抗栓作用。

LPS调控人牙龈成纤维细胞表达uPA的研究

目的 :观察LPS对牙龈成纤维细胞表达尿激酶型纤溶酶原激活剂 (uPA)的影响。方法 :采用Westernblotting和Northernblotting分别观察LPS对牙龈成纤维细胞内uPA蛋白质表达水平和mRNA表达水平的影响。结果 :经 1.0 μg/mL LPS处理 8h后 ,牙龈成纤维细胞内uPA蛋白表达量明显增强 ,且这一增强作用可持续 2 4h ;经 1.0 μg/mLLPS处理 4h后 ,牙龈成纤维细胞内uPAmRNA表达量明显增强。 结论 :LPS能够促进牙龈成纤维细胞表达uPA ,参与牙周组织的破坏。

P12-LOX和uPA在人乳腺癌中的表达及其相关性

目的:探讨血小板型12脂氧合酶(P12-LOX)和尿激酶型纤溶酶原激活物(u PA)在人乳腺癌中的表达及与乳腺癌侵袭、转移的关系。方法:应用免疫组化方法检测24例三阴性乳腺癌、58例非三阴性乳腺癌和20例乳腺良性疾病组织标本中P12-LOX和u PA的表达情况,并分析其相关性及与临床病理特征进行比较。运用RT-PCR的方法检测三种不同侵袭能力乳腺癌细胞株中P12-LOX和u PA的mRNA表达情况并分析其与乳腺癌侵袭转移的关系。结果:u PA和P12-LOX蛋白在三种乳腺癌组织中的阳性表达率皆呈现出随着肿瘤恶性程度增高而逐渐增高的趋势,统计结果显示具有显著统计学意义(P<0.01);在乳腺癌组织中u PA、P12-LOX蛋白表达之间存在显著正相关(r=0.512,P<0.01)。u PA、P12-LOX表达与临床分期、淋巴结转移显著相关(P<0.01)。与患者的年龄、肿瘤大小无明显相关性(P>0.05)。在MDA-MB-231细胞系、MCF-7细胞系、HBL-100细胞系中P12-LOX和u PA的mRNA的表达随细胞侵袭能力的增强表达增高,各组间有显著统计学差异(P<0.01)。结论:乳腺癌组织中存在u PA和P12-LOX的高表达,且与乳腺癌的侵袭转移相关,提示两者可作为乳腺癌预后不良的指标之一。

uPA-uPAR系统对骨巨细胞瘤细胞p44(MAPK)信号转导的影响

目的研究uPA/uPAR系统对骨巨细胞瘤细胞MAPK信号转导的影响。方法分离并培养骨巨细胞瘤细胞,用免疫组化检测骨巨细胞瘤组织中uPAR的表达水平,用免疫共沉淀方法检测外源激活或阻断uPA-uPAR对骨巨细胞瘤细胞信号转导通路的p44(MAPK)蛋白磷酸化水平的影响。结果仅有单核基质细胞可以在体外培养中长期生存;在骨巨细胞瘤组织中uPAR主要表达在部分单核基质细胞和一些多核巨细胞的胞膜上;将uPA-ATF加入培养的骨巨细胞瘤细胞后,细胞信号通路上的p44蛋白磷酸化水平明显增高。用uPAR抗体处理后,细胞p44蛋白磷酸化水平明显降低。说明uPA-ATF具有细胞信号转导活性,该活性受uPAR拮抗剂的影响。结论本实验检测到uPA-uPAR系统是通过p44(MAPK)信号转导通路转导信息,从而调节骨巨细胞瘤细胞增殖、分化及其他生物学行为。