New developments in the urokinase-type plasminogen activator system

The urokinase-type plasminogen activator (uPA) system plays a central role in control of cell surface proteolysis and extracellular matrix degradation. Components of this system are upregulated in a wide variety of human cancers and high levels of these proteins predict more rapid relapse and shorter survival. Recently, additional complexities in this system have been recognised, especially with regard to the roles of plasminogen activator inhibitor-1 (PAI-1), the urokinase receptor (uPAR) and urokinase:uPAR complexes. PAI-1 has been shown to play a major role in the process of pathological angiogenesis. The uPAR is involved as a key player both in proteolysis and cellular adhesion, where it is both an adhesion receptor itself for vitronectin and interacts with and modifies signalling from integrins. In addition, binding of uPA to the receptor can induce intracellular signalling via a number of different pathways, including integrins and G proteins. These new developments lead to a number of novel targets for drug discovery beyond better established enzyme inhibitors and receptor antagonists.     

Quantitative DNA methylation analysis of laser capture microdissected formalin-fixed and paraffin-embedded tissues

We developed an assay to quantify DNA methylation in breast cancer cells isolated by laser capture microdissection (LCM). The assay uses methylation sensitive restriction enzyme (MSRE) digestion and quantitative polymerase chain reaction (qPCR). To assess the validity and precision of the assay, we prepared standard samples with expected methylation percentage (MP) for two gene promoters (PLAU (plasminogen inhibitor, urokinase) and TIMP3 (TIMP metallopeptidase inhibitor 3)) that we compared with measured MPs. We found good linearity of MSRE digestion and qPCR procedures for both promoters (β = 0.90-1.19 ± 0.05-0.10 and r = 0.95-0.98; all P < 0.0001). Moreover, results remained similar after addition of a purification step between MSRE digestion and qPCR procedures. The validity of this technique was also confirmed by successfully replicating previously published MPs of four cell lines for PLAU and TIMP3 promoters. We assessed the consistency of our approach by comparing MPs of PLAU and TIMP3 promoters from nine breast cancer patients and two cell lines using LCM frozen tissues and their corresponding formalin-fixed paraffin-embedded tissues. We found good consistency (intraclass correlation coefficient = 0.93) of MPs between frozen tissues and formalin-fixed paraffin-embedded tissues. Our data demonstrate that this assay based on digestion with MSRE and qPCR procedures is a good technique to quantify MP on limited amounts of DNA and may find clinical applications.     

Fourier analysis of corneal astigmatic changes following photorefractive keratectomy

The purpose of this study was to evaluate the corneal irregular astigmatism following photorefractive keratectomy (PRK) for myopia. The corneal topography of 30 eyes of 26 patients was measured with the TMS-1 videokeratoscope before and 1 month after PRK. Axial dioptric data were decomposed into four components; A0 (Sphericity), C1 × 2 (Asymmetry), C2 × 2 (Regular astigmatism), and C3 (higherorder irregularity) for the central 3 and 6 mm zone by Fourier series harmonic analysis. Post-operative topographies were divided into those with an irregular and those with a homogeneous pattern, and the Fourier components were compared. In the 6mm zone, A0 was significantly decreased (P &lt; 0.001), and C1 × 2, C2 × 2, and C3 were significantly increased (P = 0.001, 0.005, 0.002, respectively). In the 3mm zone, A0 decreased (P &lt; 0.001) and C1 × 2 increased (P &lt; 0.001) significantly. C1 × 2 was correlated with the post-operative corrected visual acuity (P &lt; 0.001, r = 0.647). The irregular pattern group had a larger C1 × 2 component (P &lt; 0.001). The treatment displacement was not correlated with any component. In conclusion, irregular topography due to intraoperative drift or asymmetrical wound healing may play a more important role in the post-operative corneal optical property than mild treatment displacement.     

尿激酶治疗结核性包裹性胸膜炎88例

目的评价胸腔内注入尿激酶对治疗结核性包裹性胸膜炎的疗效。方法88例结核性包裹性胸膜炎随机分成治疗组与对照组,治疗组每次抽液后注入尿激酶10万单位+地塞米松10mg+生理盐水20ml,对照组抽液后,注入地塞米松10mg+生理盐水20ml,其他治疗相同。结果治疗组胸水引流量明显增加,胸水吸收时间明显优于对照组,遗留胸膜粘连及肥厚明显少于对照组,且无并发症发生。结论胸腔内注入尿激酶显著增加胸液收流量,能有效降低胸膜粘连和肥厚的机会和程度。     

Preanalytical aspects of routine coagulation measurements

Pretreatment of native plasminogen (plg) with plasmin or activators resulted in an increase in the rate of activation (RA) by urokinase and a shift in mobility from β₂ to γ in agarose gel electrophoresis at pH 8.6. 6-Aminohexanoic acid (6-AHA) had the same effects at c=10^-4 to 3.3 ± 10^-3 mol/1, probably owing to a reversible binding to pig. 6-AHA produced a decrease in RA at higher c, probably owing to a reversible binding to urokinase. The high RA of pretreated pig was inhibited at c > 10^-4 mol/1. L-Lysine and trans-4-aminomethylcyclohexane-1-carboxylic acid had the same effects as 6-AHA on RA and mobility at 7 times higher and lower c. Dodecyl sulphate polyacrylamide electrophoresis showed that pretreated pig had a lower Mr than native plg (δMr 3700).     

H-Ras increases urokinase expression and cell invasion in genetically modified human astrocytes through Ras/Raf/MEK signaling pathway

Previous study reported that the activation of Ras pathway cooperated with E6/E7-mediated inactivation of p53/pRb to transform immortalized normal human astrocytes (NHA/hTERT) into intracranial tumors strongly resembling human astrocytomas. The mechanism of how H-Ras contributes to astrocytoma formation is unclear. Using genetically modified NHA cells (E6/E7/hTERT and E6/E7/hTERT/Ras cells) as models, we investigated the mechanism of Ras-induced tumorigenesis. The overexpression of constitutively active H-RasV12 in E6/E7/hTERT cells robustly increased the levels of urokinase plasminogen activator (uPA) mRNA, protein, activity and invasive capacity of the E6/E7/hTERT/Ras cells. However, the expressions of MMP-9 and MMP-2 did not significantly change in the E6/E7/hTERT and E6/E7/hTERT/Ras cells. Furthermore, E6/E7/hTERT/Ras cells also displayed higher level of uPA activity and were more invasive than E6/E7/hTERT cells in 3D culture, and formed an intracranial tumor mass in a NOD-SCID mouse model. uPA specific inhibitor (B428) and uPA neutralizing antibody decreased uPA activity and invasion in E6/E7/hTERT/Ras cells. uPA-deficient U-1242 glioblastoma cells were less invasive in vitro and exhibited reduced tumor growth and infiltration into normal brain in xenograft mouse model. Inhibitors of Ras (FTA), Raf (Bay 54-9085) and MEK (UO126), but not of phosphatidylinositol 3-kinase (PI3K) (LY294002) and of protein kinase C (BIM) pathways, inhibited uPA activity and cell invasion. Our results suggest that H-Ras increased uPA expression and activity via the Ras/Raf/MEK signaling pathway leading to enhanced cell invasion and this may contribute to increased invasive growth properties of astrocytomas.     

Thrombolytic evacuation of post-craniotomy epidural haematomas using closed suction drains: a pilot study

Summary Background. As an effective treatment for post-craniotomy epidural haematomas (EDHs), a novel method of urokinase instillation using a closed suction drain is presented and the procedure feasibility and outcomes assessed. Method. A closed system, comprising a closed suction drain with a three-spring 200 mL evacuator, fluid bag with urokinase, and syringe, was constructed to instill urokinase and evacuate a postoperative EDH. Nine patients with a symptomatic, localised EDH under a bone flap after a craniotomy underwent successive urokinase instillation following the proposed protocol. Measurement of the EDH volume and clinical evaluation were performed. Findings. An improvement of computerised tomography findings and clinical state after urokinase instillation was observed in all patients. Six urokinase instillations lasting 12 h in 6 patients with an EDH (18.2 ± 2.4 mL) and 12 urokinase instillations lasting 24 h in the other 3 patients with an EDH (33.0 ± 7.9 mL) succeeded in achieving a minimal residual EDH (6.1 ± 2.8 mL). The EDH volume decreased at a rate of 13.0 ± 2.3 mL/12 h. The GCS scores increased immediately after thrombolytic evacuation of the EDHs in 6 out of the 9 patients. For the other three patients who did not show a change of GCS score, the severe headaches were improved. All the patients were successfully treated using the proposed technique with no procedural complications such as haemorrhage or infection in the operative wound. Conclusions. This pilot study demonstrated that thrombolytic evacuation of a post-craniotomy EDH using a closed suction drain is feasible without complications and may be associated with better outcomes. Correspondence: Jaechan Park, M.D., Department of Neurosurgery, Kyungpook National University Hospital 50, Samduk 2-ga, Jung-gu, Daegu, Republic of Korea.     

uPA系统与神经胶质瘤局部侵袭

uPA系统包括尿激酶型纤溶酶原激活物（urokinase plasminogen activator, uPA）、 尿激酶型纤溶酶原激活物受体（urokinase plasminogen activation receptor, uPAR）和纤溶酶原激活物抑制剂(plasminogen activator inhibitor, PAI),它们参与了多种人类恶性肿瘤的局部侵袭和转移,是目前肿瘤治疗重要的分子靶点。uPA能激活纤溶酶原降解细胞外基质与基底膜,uPAR则能显著提升uPA的激活纤溶酶原的功能,两者结合能够增强肿瘤的侵袭性与转移能力。PAI主要通过促进血管内皮生长因子（vascular endothelial growth factor,VEGF）的表达,促进新生血管的形成,从而促进肿瘤的局部侵袭,但PAI又可通过抑制uPA-uPAR复合体的生物学活性来抑制肿瘤的局部侵袭。神经胶质瘤是最常见的颅内肿瘤,很少转移到颅外,局部侵袭是其预后不良的主要原因。近年来发现uPA系统的表达水平与神经胶质瘤的恶性程度呈正相关。本文综述了uPA系统对神经胶质瘤局部侵袭的影响及其在治疗中的意义。     

房颤对急性缺血性脑卒中患者尿激酶溶栓疗效的影响

目的 探讨房颤是否对急性缺血性脑卒中患者尿激酶溶栓疗效产生影响及对于合并房颤的急性缺血性脑卒中患者是否给予尿激酶溶栓治疗.方法 本研究为回顾性病例对照研究.从2006年4月到2012年1月连续收集发病6小时内给予尿激酶溶栓的急性缺血性脑卒中患者作为研究对象.根据有无合并房颤将符合入选标准的病例分为两组:房颤组(26例)和无房颤组(60例).采用美国国立卫生研究院卒中量表(NIHSS)、改良的Rankin量表评价治疗效果.结果 房颤组与无房颤组溶栓治疗后7d溶栓有效率比较,差异无统计学意义(57.7％ vs 56.7％,P＞0.05).在尿激酶静脉溶栓治疗后90 d,房颤组57.7％的病人功能恢复好,无房颤组65.0％的病人功能恢复好,两组比较差异无统计学意义(P＞0.05).房颤组颅内出血的发生率、症状性颅内出血的发生率及死亡率均较高,但与无房颤组比较差异均无统计学意义.结论 合并房颤的急性缺血性脑卒中患者与无合并房颤的急性缺血性脑卒中患者均可以从溶栓中获益,房颤对急性缺血性脑卒中患者尿激酶溶栓疗效无显著影响,合并房颤的急性缺血性脑卒中患者应予尿激酶溶栓治疗.