神经营养素对小胶质细胞分泌的血浆蛋白溶酶原及其激活因子的调节作用

目的探讨神经营养素对培养的小胶质细胞分泌的血浆纤维蛋白溶酶原（ＰＧｎ）及其激活因子（ｕＰＡ）的调节作用。方法利用神经营养素［神经生长因子（ＮＧＦ）、脑源神经营养因子（ＢＤＮＦ）、神经营养素３（ＮＴ－３）和营养素４（ＮＴ－４）］对体外培养的大鼠脑小胶质细胞进行刺激，然后采用酶谱分析法、免疫印迹法及免疫细胞化学分析法对其产生的ＰＧｎ和ｕＰＡ进行测定。结果实验所用的神经营养素对所测定的酶原及其激活因子皆有上调作用。结论在体外，神经营养素调节小胶质细胞的功能     

Involvement of protein kinase C in glutamate release from cultured microglia

Glutamate release from microglial cells may cause neuronal damage. To elucidate the mechanism of glutamate release, we examined the possible regulation by nitric oxide and protein kinase C. Cultured microglia prepared from the whole brains of newborn rats released glutamate by the stimulation with lipopolysaccharide (LPS) dose dependently. The time course study revealed that glutamate release showed a long lag time about 6 h after LPS stimulation, whereas about 3 h lag time was observed in LPS-induced NO production. An inhibitor for NO synthase, N(G)-nitro-L-arginine, could effectively inhibit the glutamate release. Glutamate release induced by LPS was enhanced by 1 nM phorbol myristate acetate (PMA). Furthermore, high concentrations of PMA (>10 nM) induced glutamate release even without LPS stimulation. Glutamate release stimulated either by 100 ng/ml LPS or 100 nM PMA was inhibited by staurosporine, and also by alpha-aminoadipate. These results provide insight into the pathways regulating microglial pathological activation by protein kinase C and may be a base for the protection against microglia-evoked neurotoxicity.     

DDAVP induces systemic release of urokinase-type plasminogen activator

The desamino-d-arginine vasopressin (DDAVP) induced enhancement of endogenous fibrinolysis is generally attributed to the release of tissue-type plasminogen activator (t-PA) from the vessel wall. The observation of concurrent release of urokinase-type plasminogen activator (u-PA), which eventually might cooperate in the enhanced fibrinolytic activity, has not been reported thus far. In a preliminary study in two healthy human volunteers we found a 1.8-fold increase of urokinase-antigen (UK-antigen) and a 1.7-fold increase of plasmin-activatable pro-urokinase (pro-UK) activity to DDAVP intravenously. The plasma-peak levels coincided with the maximal t-PA level. These responses following infusion of DDAVP were subsequently confirmed in a randomized double blind cross-over study in six human volunteers. We conclude that u-PA is released by DDAVP concurrently with t-PA and that it is presumably from the same origin as t-PA i.e. endothelial cells. u-PA and t-PA may therefore cooperate in the enhanced fibrinolytic activity upon DDAVP infusion.     

Half-life of single-chain urokinase-type plasminogen activator (scu-PA) and two-chain urokinase-type plasminogen activator (tcu-PA) in patients with acute myocardial infarction

The pharmacokinetics of urokinase (two-chain urokinase-type plasminogen activator, tcu-PA) and single-chain urokinase-type plasminogen activator (scu-PA) were studied in 20 patients with acute myocardial infarction (AMI). Ten consecutive patients received 2.5 million units tcu-PA by bolus injection within 5 min during the first 6 h after AMI (group I). Ten further consecutive patients received 250,000 U tcu-PA within 5 min, followed by 4.5 million U scu-PA by intravenous infusion over 40 min (group II). An enzyme immunoassay was developed for urokinase antigen determinations, and a fibrin plate assay for determinations of fibrinolytic activity was applied. Using a 3-compartment model, in group I 98% of urokinase antigen were cleared with a half-life of 60.8 min. After scu-PA, urokinase antigen was cleared with half-lives (area under the curve in parentheses) of 6.9 min (74.8%), 26.5 min (23.6%), and 329.7 min (2.2%). The half-disappearance times of fibrinolytic activity were 18 and 8 min in group I and II, respectively. A more pronounced decrease of plasminogen was observed after tcu-PA.     

Direct interaction of the kringle domain of urokinase-type plasminogen activator (uPA) and integrin alpha v beta 3 induces signal transduction and enhances plasminogen activation

It has been questioned whether there are receptors for urokinase-type plasminogen activator (uPA) that facilitate plasminogen activation other than the high affinity uPA receptor (uPAR/CD87) since studies of uPAR knockout mice did not support a major role of uPAR in plasminogen activation. uPA also promotes cell adhesion, chemotaxis, and proliferation besides plasminogen activation. These uPA-induced signaling events are not mediated by uPAR, but mediated by unidentified, lower-affinity receptors for the uPA kringle. We found that uPA binds specifically to integrin alpha v beta 3 on CHO cells depleted of uPAR. The binding of uPA to alpha v beta 3 required the uPA kringle domain. The isolated uPA kringle domain binds specifically to purified, recombinant soluble, and cell surface alpha v beta 3, and other integrins (alpha 4 beta 1 and alpha 9 beta 1), and induced migration of CHO cells in an alpha v beta 3-dependent manner. The binding of the uPA kringle to alpha v beta 3 and uPA kringle-induced alpha v beta 3-dependent cell migration were blocked by homologous plasminogen kringles 1-3 or 1-4 (angiostatin), a known integrin antagonist. We studied whether the binding of uPA to integrin alpha v beta 3 through the kringle domain plays a role in plasminogen activation. On CHO cell depleted of uPAR, uPA enhanced plasminogen activation in a kringle and alpha v beta 3-dependent manner. Endothelial cells bound to and migrated on uPA and uPA kringle in an alpha v beta 3-dependent manner. These results suggest that uPA binding to integrins through the kringle domain plays an important role in both plasminogen activation and uPA-induced intracellular signaling. The uPA kringle-integrin interaction may represent a novel therapeutic target for cancer, inflammation, and vascular remodeling.     

尿激酶型纤溶酶原激活剂在人脑胶质瘤中的表达

目的　探讨尿激酶型纤溶酶原激活剂 (uPA)在人脑胶质瘤中的表达特征 ,研究其表达的临床意义。方法　用免疫组化的方法检测uPA在 5 2例胶质瘤、4例垂体腺瘤、5例正常脑组织中的表达情况 ,并结合临床资料分析其表达的意义。结果　 5 2例胶质瘤中均有uPA的表达 ,随着其恶性程度的升高 ,其表达也随之升高 ,且分布不均匀。在垂体腺瘤中低度表达 ,在正常脑组织中无表达。高级别胶质瘤中uPA的表达与低级别胶质瘤比较有显著差异性 (P <0 .0 1) ;低级别胶质瘤与垂体腺瘤和正常脑组织中uPA的表达也有显著差异性 (P <0 .0 1)。生存期 <3年者uPA的表达显著高于生存期 >3年者 (P <0 .0 1)。结论　uPA的表达与胶质瘤的侵袭性和血管的生成等有关 ,并对其预后的判断有一定意义。     

Potentiation by Lys-plasminogen of clot lysis by single or two chain urokinase-type plasminogen activator or tissue-type plasminogen activator

Study has been made of the influence of addition of human NH2 terminal glutamic acid plasminogen (Glu-Plg) or human NH2 terminal lysine plasminogen (Lys-Plg) to normal citrated plasma upon the rate of lysis of fully crosslinked plasma clots in the presence of single or two chain urokinase type plasminogen activator (scu-PA/tcu-PA) or tissue plasminogen activator (t-PA). The specificity of any thrombolytic property was evaluated by measurement of plasma fibrinogen levels. Lys-Plg added to a concentration of 20% of normal plasma plasminogen caused 5 to 6 fold increase in the extent of lysis observed at 6 hours by 100 units/ml of scu-PA and with a small increase in fibrinogenolysis. Glu-Plg added at 20% of normal level had no influence on thrombolysis but at 50% of normal caused increased thrombolysis with rapid depletion of plasma fibrinogen. An apparently synergistic effect of addition of tcu-PA on scu-PA activity was increased by addition of plasminogen (e.g. addition of 20% Lys-Plg increased the lysis rate 4 to 5 fold over the first hour equivalent to an increase of potency of approximately three to four fold). Addition of plasminogen up to double the normal plasma concentration was observed to have no influence on clot lysis in the presence of t-PA. Plasminogen potentiated the rate of lysis by scu-PA/t-PA synergic mixtures with an approximately 1.5 to 1.9 fold increase in potency. Potentiation occurred without increase in the depletion of plasma fibrinogen.(ABSTRACT TRUNCATED AT 250 WORDS)     

Physical exercise induces enhancement of urokinase-type plasminogen activator (u-PA) levels in plasma

The enhancement of the blood fibrinolytic potential by physical exercise is generally attributed to the release of tissue-type plasminogen activator (t-PA) from the vessel wall. In this study we have investigated the possible contribution of urokinase-type plasminogen activator (u-PA). Six healthy male volunteers (age 21-25 years) were screened for their ability to perform maximal exercise for their age-group for 12 min on a bicycle ergometer. Subsequently, on one occasion they were required to remain supine for 2 h (from 8.30 a.m. onwards) an on another they performed maximal exercise (from 9.00 a.m. onwards). During exercise an increase in u-PA antigen and plasmin-activatable pro-urokinase (proUK) activity, concurrent with t-PA antigen and euglobulin t-PA activity, was observed in all six volunteers, while at rest these parameters remained unaffected. Mean u-PA- and t-PA antigen increased, respectively, from 4.2 +/- 1.0 ng/ml and 5.8 +/- 2.1 ng/ml before exercise to 9.8 +/- 3.0 ng/ml and 18.3 +/- 3.8 ng/ml (peak). Mean plasmin-activatable proUK activity and t-PA activity increased, respectively, from 2.1 +/- 0.4 ng/ml and 0.3 +/- 0.2 ng/ml before exercise to 4.3 +/- 1.7 ng/ml and 7.2 +/- 4.0 ng/ml (peak). The increases were statistically significant throughout (paired t-test, pre vs post, antigen P less than 0.005 and activity P less than 0.02). After cessation of exercise u-PA and t-PA declined concurrently to normal values with a 50% decay in about 5 min.(ABSTRACT TRUNCATED AT 250 WORDS)     

Beta-adrenergic receptor mediated release of tissue plasminogen activator in anaesthetized dogs

Drugs like epinephrine or isoproterenol can enhance fibrinolytic activity of blood. It is still a matter of debate whether this effect can be totally or only partially blocked by beta-receptor blockers. We have developed a dog model for ex vivo measurement of the fibrinolytic activity using a spectrophotometric assay employing human plasminogen, chromogenic plasmin substrate and human fibrinogen-BrCN digests for the stimulation of t-PA. After i.v. administration of isoproterenol the fibrinolytic activity increased, but this could be seen only in the presence of fibrinogen-BrCN digest in the test system. This suggests that isoproterenol caused higher levels of dog t-PA. Pretreatment with the beta-blocker propranolol completely blocked the increase in t-PA.     

The complex between urokinase-type plasminogen activator (uPA) and its type-1 inhibitor (PAI-I) independently predicts response to first-line endocrine therapy in advanced breast cancer

It has been shown that urokinase-type plasminogen activator (uPA) and its main inhibitor (PAI-I) have predictive value for therapy success in advanced breast cancer. Levels of the complex between uPA and PAI-I, formed when both molecules are in their active form, might have superior predictive power. Here, we investigate the association between levels of uPA:PAI-I complex and rate of response to first-line systemic therapy for advanced breast cancer. Tumor tissues of 170 patients with advanced breast cancer were analyzed for uPA:PAI-I complex concentrations using a quantitative enzyme-linked immunosorbent assay. The patients received either endocrine therapy (n=96) or chemotherapy (n=74) as first-line treatment after diagnosis of advanced disease. Of the endocrine treated patients, those with high levels of uPA:PAI-I complex showed a shorter progression-free survival (PFS) compared to patients with lower uPA:PAI-I complex levels (P=0.035). Furthermore, in the multivariate regression analysis a significant lower rate of response to first-line endocrine therapy was found in patients with high uPA:PAI-I complex levels compared to patients with low uPA:PAI-I complex levels (odds ratio (OR)=0.27, 95% CI, 0.09-0.59, P=0.018), in addition to the predictive impact of the steroid hormone receptor (ER/PgR) status (OR=2.68, 95% CI, 1.08-6.63, P=0.033). Complex levels did not predict efficacy of chemotherapy in patients with advanced breast cancer. The results show that the plasminogen activation system affects the response to endocrine therapy independent of steroid hormone receptor status and may be of help to further refine the indication for this treatment in individual patients. Further studies are warranted to explain this underlying resistance to endocrine therapy when uPA:PAI-I levels are high.     

Enhanced release of plasminogen activator inhibitor(s) but not of plasminogen activators by cultured rat glial cells treated with interleukin-1

Astroglial cells are known to proliferate during development of the nervous system, as well as during post-traumatic gliosis. We have previously shown that the proliferation of cultured astrocytes can be stimulated by the urokinase-type (uPA) of plasminogen activator (PA) and that astrocytes are able to release such uPA upon stimulation with basic fibroblast growth factor, which is known to act as a mitogen for these cells. Here we report studies on the effects of human interleukin-1 (IL-1) on the release of PA activity by cultured newborn rat astroglial cells. Whereas there is controversy in the literature as to whether IL-1 stimulates multiplication of astroglial cells, we failed to observe such an effect in our system. We did observe, however, a dose-dependent decrease in PA activity in the supernatant of the IL-1 treated cultures. Further analysis revealed that this apparent decrease in PA release was in fact due to an increased release of plasminogen activator inhibitor (PAI). A similar IL-1 induced increase in PAI release was also found to occur in cultures of transformed astrocytes (human glioma LN18) and in cultured Schwann cells, but not in cultures of neurons or neuronal tumour cells. Since protease inhibitors are known to possess neuritogenic properties, our results suggest that IL-1, by its capacity to induce PAI, may promote neuritogenesis.     

Differential detection of single-chain and two-chain urokinase-type plasminogen activator by a new immunoadsorbent-amidolytic assay (IAA)

A new immunoadsorbent-amidolytic assay (IAA) for the specific differential detection of two-chain urokinase-type plasminogen activator (tcu-PA) and its single-chain precursor (scu-PA) in cell culture supernatants has been developed. The assay combines the selectivity of immunoassays with the specificity of enzyme activity assays exploiting both the antigenic and enzymatic properties of the two proteins. tcu-PA and scu-PA are selectively immunoadsorbed on the wells of a microtiterplate coated with the monoclonal antibody 5B4 and tested for enzymatic activity before and after activation by plasmin treatment. Both proteins are determined with similar efficiency since overlapping dose-response curves were obtained in the range between 12.5-200 ng/ml. The assay has been used to determine tcu-PA and scu-PA in A431 human epidermoid carcinoma cell supernatants. The analytical recoveries for tcu-PA and scu-PA added to A431 cell supernatants were 95.2% and 96.9% respectively. The intra- and inter-assay variations (CV) were 5.5% and 9.0% for tcu-PA and 9.7% and 9.8% for scu-PA respectively.     

Absence of synergism between tissue-type plasminogen activator (t-PA), single-chain urokinase-type plasminogen activator (scu-PA) and urokinase on clot lysis in a plasma milieu in vitro

A potential synergic effect of tissue-type plasminogen activator (t-PA), single-chain urokinase-type plasminogen activator (scu-PA) or urokinase on clot lysis was investigated in a whole human plasma system in vitro. The system consisted of a human plasma clot labeled with 125I-fibrinogen, immersed in citrated whole human plasma, to which the thrombolytic agents were added. Clot lysis was quantitated by measurement of released 125I, and activation of the fibrinolytic system in the surrounding plasma by measurements of fibrinogen and alpha 2-antiplasmin. t-PA, scu-PA and urokinase induced a dose-dependent and time-dependent clot lysis; 50 percent lysis after 2 h was obtained with 5 nM t-PA, 20 nM scu-PA and 12 nM urokinase. At these concentrations no significant activation of the fibrinolytic system in the plasma was observed with t-PA and scu-PA, whereas urokinase caused significant alpha 2-antiplasmin consumption and concomitant fibrinogen degradation. The shape of the dose-response curves was different; t-PA and urokinase showed a log linear dose-response whereas that of scu-PA was sigmoidal. Combinations of t-PA and scu-PA, of t-PA and urokinase or of scu-PA and urokinase at thrombolytic doses of each showed no synergism for thrombolysis. Fifty percent clot lysis in 2 h was obtained at total concentrations of the combined agents of 5 to 15 nM with molar ratios ranging from 1:4 to 4:1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and characterization of single-chain urokinase-type plasminogen activator (pro-urokinase) from human A431 cells

A single-chain urokinase-type plasminogen activator (A431sc-uPA) was purified approximately 18,000-fold from A431 human epidermoid carcinoma cell supernatants by monoclonal antibody immunoaffinity chromatography on 5B4-agarose and ion-exchange FPLC (overall yield 63%). More than 100 micrograms of A431sc-uPA can be recovered per liter of supernatant. The product is homogeneous by SDS-PAGE and reverse phase FPLC analysis while two main isoelectric forms of pI 9.05 and pI 9.20 were observed by IEF. SDS-PAGE in reducing and non-reducing conditions, Western blot analysis and zymography showed that A431sc-uPA is a single-chain protein of about 50,000 Mr immunologically related to urokinase (uPA) and distinct from tissue plasminogen activator (tPA). The N-terminal aminoacid sequence of A431sc-uPA (27 residues) is identical to that of human kidney single-chain uPA. A431sc-uPA does not incorporate 3H-diisopropylfluorophosphate and is virtually inactive on the synthetic substrate S-2444. Plasmin treatment converts A431sc-uPA into a two-chain active form with a fibrinolytic specific activity of 123,000 I. U./mg.     

Synergistic effect on thrombolysis of sequential infusion of tissue-type plasminogen activator (t-PA) single-chain urokinase-type plasminogen activator (scu-PA) and urokinase in the rabbit jugular vein thrombosis model

In a quantitative model of thrombolysis, consisting of rabbits with a 125I-fibrin labeled blood clot in the jugular vein, simultaneous intravenous infusion over 4 hours of t-PA and scu-PA or of t-PA and urokinase had a significantly greater (p less than 0.01) thrombolytic effect than could be anticipated on the basis of the added effects of each agent alone. In order to further investigate the mechanism of this in vivo synergism, recombinant t-PA (rt-PA) and scu-PA in synergistic amounts were infused: 1) simultaneously over 4 hours, 2) rt-PA over 1 hour, then 15 min later scu-PA over 2 hours and 3) scu-PA over 1 hour, than 15 min later rt-PA over 2 hours. Simultaneous infusion of 0.1 mg/kg rt-PA and 0.2 mg/kg scu-PA gave 48 +/- 2 percent thrombolysis (mean +/- SEM, n = 5) and of 0.2 mg/kg rt-PA and 0.4 mg/kg scu-PA 67 +/- 5 percent (n = 5). When these infusions were given sequentially, rt-PA followed by scu-PA gave 32 +/- 5 (n = 4) and 49 +/- 8 (n = 4) percent lysis, but scu-PA followed by rt-PA yielded only 14 +/- 1 (n = 4) and 21 +/- 1 (n = 4) percent lysis, indicating that synergism occurs when rt-PA is followed by scu-PA but not when scu-PA is followed by rt-PA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pro-urokinase up-regulates the expression of urokinase-type plasminogen activator (u-PA) in human pulmonary arterial endothelial cells

The fibrinolytic function of endothelial cells plays an important role in the pathophysiology of pulmonary vascular diseases. In this study, the effects of pro-urokinase, a new thrombolytic drug that is currently being tested for the treatment of pulmonary embolism, on the expression of urokinase-type plasminogen activator (u-PA) and u-PA receptor (u-PAR) were assessed. The role of u-PAR was also investigated. Immunocytofluorescence and RT-PCR techniques were employed. In normal human pulmonary arterial endothelial cells (HPAECs), the expression levels of u-PA and u-PAR were very low. Incubation with pro-urokinase up-regulated u-PA expression at both the mRNA level and the protein level; however, the expression of u-PAR was not affected. The effect of pro-urokinase induction was totally inhibited by the release of u-PAR from the HPAECs' surface with PLC. This result suggests that the combination of u-PA with u-PAR may be a critical pathway for the induction of u-PA expression.     

Impairment of adipose tissue development by hypoxia is not mediated by plasminogen activator inhibitor-1

Hypoxia in rodents and humans is associated with a reduction of body fat on the one hand, and with enhanced expression of plasminogen activator inhibitor-1 (PAI-1), the main inhibitor of the fibrinolytic system, on the other hand. It was the objective of this study to investigate whether impairment of adipose tissue development by hypoxia may be mediated by PAI-1. Five week old male wild-type (WT) C57Bl/6 mice were fed a standard (SFD) or high fat (HFD) diet and kept under normoxic or hypoxic (10% O(2)) conditions. In addition, PAI-1 deficient mice and WT littermates were kept on HFD under normoxia or hypoxia. In vitro, the effect of hypoxia (2% O(2)) was investigated on differentiation of 3T3-L1 cells into adipocytes. Hypoxia induced a significant reduction of weight gain in WT mice on either SFD or HFD, accompanied by lower weights of subcutaneous (SC) and gonadal (GON) fat. Under hypoxic conditions, adipocytes in the adipose tissues were significantly smaller, whereas blood vessel size and density were larger. Serum PAI-1 levels were enhanced in hypoxic mice on SFD but not on HFD, and overall did not correlate with the observed changes in adipose tissue composition. Furthermore, the effects of hypoxia on adipose tissue in mice on HFD were not affected by deficiency of PAI-1. The inhibiting effect of hypoxia on in vitro preadipocyte differentiation was not mediated by PAI-1 activity. In conclusion, impairment of in vivo adipose tissue development and in vitro differentiation of preadipocytes by hypoxia is not mediated by PAI-1.