Breast cancer cell surface annexin II induces cell migration and neoangiogenesis via tPA dependent plasmin generation

Annexin II, an abundant phospholipids binding cell surface protein, binds tPA and functions as a regulator of fibrinolysis. Annexin II also mediates angiogenesis and enhances tumor growth and metastasis. However, the mechanism supporting this role is not known. Using human breast cancer model we show that invasive human breast cancer cells (MDA-MB231) synthesize annexin II and tissue plasminogen activator (tPA). In vitro both annexin II and tPA interacts which in turn convert zymogen plasminogen to reactive enzyme plasmin. Cell surface produced plasmin inhibited the migration of MDA-MB231 cells. Silencing of annexin II gene in MDA-MB231 cells abolished tPA binding therefore inhibited tPA dependent plasmin generation. These annexin II suppressed MDA-MB231 cells showed reduced motility. Immunohistochemical analysis of prediagnosed clinical specimens showed abundant secretion of tPA and expression of annexin II on the surface of invasive human breast cancer cells which correlates with neovascularization of the tumor. Taken together, these data indicate that annexin II may regulate localized plasmin generation in breast cancer. This may be an early event switching breast cancer from the prevascular phase to the vascular phase and thus contributing to aggressive cancer with the possibility of metastasis. The data provide a mechanism explaining the role of annexin II in breast cancer progression and suggest that annexin II may be an attractive target for therapeutic strategies aimed to inhibit angiogenesis and breast cancer.     

凝血、纤溶系统功能与2型糖尿病关系的研究

目的探讨机体凝血和纤溶系统功能的改变与2型糖尿病并发症的关系。方法选取49例2型糖尿病患者，按其24h内尿白蛋白量分为尿白蛋白正常组21例，微白蛋白尿组15例，临床肾病组7例，另为合并急性并发症组6例。同时选择30例年龄与患者相匹配的健康者作为对照组。测定血浆组织型纤溶酶原激活物（tPA）含量和纤溶酶原激活物抑制剂（PAI）含量、纤溶酶原（PLG）活性、D-二聚体（D-D）含量、凝血酶激活的纤溶抑制物（TAFI）含量及其活性（TAFIa）。结果糖尿病（DM）患者tPA为14．6±5．9μg/ml和PLG94．4±14．3％，皆明显低于对照组（两者分别为17．9±6．0μg/ml，101．4±11．7％；P〈0．05）；DM组PAI33．2±11．1μg/ml、D—D0．74±0．11μg／ml、TAFI 122．36±47．1％及TAFIa77．3±8．5μg/ml，显著高于对照组（分别是25．8±7．9、0．21±0．57、89．2±31．1、27．2±10．3），差异有显著性（均P〈0．05）；分析DM肾病患者白蛋白尿正常组与对照组差异无显著性，其余4组与对照组差异均有显著性（P〈0．05）。对TAFI相关分析，表明其与患者UAE、PAI、D—D结果呈正相关。结论机体凝血纤溶功能的异常改变与2型糖尿病肾病关系密切，各项目的检验，可反映糖尿病微血管病的程度，从而有助于临床对患者病情与预后的评估。     

Lipopolysaccharide attenuates thrombolysis in batroxobin-induced lung vasculature fibrin deposition but not in ferrous chloride-induced carotid artery thrombus in rats: role of endogenous PAI-1

In this study, we investigated if elevation of endogenous plasminogen activator inhibitor type 1 (PAI-1) by lipopolysaccharide (LPS) can retard thrombolysis in both a rat model of lung vasculature fibrin deposition and a platelet-rich thrombus model induced by endothelial injury. By 3 h following an intravenous bolus injection of 0.5 mg/kg LPS, the plasma PAI-1 level had increased to 8 ng/ml. ¹²⁵I-labeled fibrinogen was injected intravenously followed by an injection of batroxobin. Batroxobin converts fibrinogen into insoluble fibrin, which was then deposited in the lungs within 5 min, followed by spontaneous fibrinolysis that completely cleared fibrin deposition in the lungs by 30 min. In rats pre-treated with LPS, spontaneous fibrinolysis was significantly retarded. In the endothelial injury model, topical application of FeCl₂ on the carotid artery induced an occlusive platelet-rich thrombus, which was not sensitive to endogenous thrombolysis. Exogenous tissue-type plasminogen activator (tPA) was required to recanalize the occlusive thrombus in a dose-dependent manner. Pre-treatment with LPS did not alter the dose–response curve of exogenous tPA-induced thrombolysis. These data indicate that batroxobin-induced lung vasculature fibrin deposition in rats, unlike the FeCl₂ model, is sensitive to the impact of endogenous PAI-1 on fibrinolysis.     

Changes in tissue-plasminogen activator mRNA expression following cortical ablation in the rat brain

Tissue plasminogen activator (tPA) has been used to treat acute thrombotic lesions. Roles other than the activation of fibrinolytic pathways have been suggested for tPA in the mature brain. We used the in situ hybridization technique to investigate the changes in tPA mRNA expression within the brain after cortical ablation. We found that expression of tPA mRNA started to increase diffusely in the cortex ipsilateral to the injury 6 h after ablation. This increase had become prominent 24 h after ablation. On d 5, the expression of tPA mRNA had returned to that of the control animals except for the area near the injury. We also found that administration of MK-801 before injury suppressed the increase of tPA mRNA in the ipsilateral cortex. These results suggest that the increase in tPA mRNA is likely to be mediated via activation of NMDA receptors.     

Analysis of Plasma Fibrinolysis in the Patients with Acute Cerebral Infarction

ＡｎａｌｙｓｉｓｏｆＰｌａｓｍａＦｉｂｒｉｎｏｌｙｓｉｓｉｎｔｈｅＰａｔｉｅｎｔｓｗｉｔｈＡｃｕｔｅＣｅｒｅｂｒａｌＩｎｆａｒｃｔｉｏｎＺＨＡＮＧＹｉｎｇ－ｄｏｎｇ（张颖冬）；ＬＩＵＸｉ－ｍｉｎ（刘锡民）；ＣＡＩＺｈｕａｎ（蔡转）；ＹＡＮＧＭｉｎｇ－...     

Thrombolysis in Young Adults

Obstacles to thrombolytic treatment could be unique in young stroke patients and identifying and addressing them would potentially enhance outcomes. However, it has not been well studied how age affects the time to presentation, diagnosis or treatment of acute strokes. Although studies suggest that younger patients may be more aware of stroke symptoms and signs than older ones, they may be less likely to use emergency medical services. Importantly, for strokes of similar severity, younger patients have more favorable outcomes with thrombolysis than older patients. On the other hand, young patients who experience extensive middle cerebral artery strokes are more likely to develop fatal brain edema than older patients. Current data support the use of thrombolytics for all ischemic stroke etiologies, including entities such as cervical artery dissection that are more common in young patients.     

Tissue plasminogen activator in primary afferents induces dorsal horn excitability and pain response after peripheral nerve injury

The extracellular protease cascade of plasminogen activators and plasminogen are known to regulate neuronal plasticity and extracellular matrix modification, and to be important factors involved in producing long-term potentiation in the CNS. The purpose of this study is to examine the expression of plasminogen activators in primary afferents and its role in nociceptive pathways after peripheral nerve injury. We found the induction of mRNAs for tissue type plasminogen activator (tPA) and urokinase plasminogen activator (uPA) in the rat dorsal root ganglia following sciatic nerve transection. Immunoreactivity for tPA was increased in laminae I and II of the dorsal horn and, importantly, the increase in proteolytic activity mediated by tPA was observed in the same area. As neither immunoreactivity for uPA nor uPA-mediated proteolysis was observed, we further examined the effects of tPA on dorsal horn excitability and neuropathic pain behaviour. Intrathecal injection of a specific inhibitor of tPA decreased electrical stimulation-induced Fos expression in dorsal horn neurons following axotomy, and also prevented the development of thermal hyperalgesia following partial sciatic nerve ligation. These findings suggest that the increased tPA in the dorsal horn due to mRNA expression in the dorsal root ganglia increases the dorsal horn excitability and has an important role in pain behaviour after peripheral nerve injury. The tPA-mediated hypersensitivity in dorsal horn neurons may be a novel molecular mechanism of neuropathic pain.     

Comparison of Tenecteplase with Alteplase on clinical rating scores following small clot embolic strokes in rabbits

Tenecteplase (TNK) was engineered to have increased fibrin specificity and an increased half-life compared to Alteplase. Although Tenecteplase is currently being tested in a Phase II clinical trial in acute ischemic stroke patients, little is known about the pharmacology and dose-response or therapeutic window for Tenecteplase in embolic stroke models. In the present study, we compared Tenecteplase with Alteplase on behavioral outcome in rabbits with embolic strokes. Male New Zealand white rabbits were embolized by injecting a suspension of small blood clots into the middle cerebral artery (MCA) via a catheter. The rabbit small clot embolic stroke model (RSCEM) was used for a dose-response profile analysis of Tenecteplase (0.1 mg/kg-3.3 mg/kg) and Alteplase (0.9 mg/kg-3.3 mg/kg) given intravenously 1 h following embolization. In additional studies, Tenecteplase (0.9 mg/kg) or Alteplase (3.3 mg/kg) was administered 3 (or 6) h following embolization to determine the therapeutic window for the thrombolytics. For both studies, behavioral analysis was conducted 24 h following embolization, allowing for the determination of the effective stroke dose (P50) or clot amount (mg) that produces neurological deficits in 50% of the rabbits. Using the RSCEM, a drug is considered beneficial if it significantly increases the P50 compared with the control group. The P50 of controls 24 h after embolization was 1.13 +/- 0.15 mg. Rabbits treated 1 h post-embolization with Tenecteplase (0.1, 0.25, 0.9, 1.5 or 3.3 mg/kg) had P50 values of 1.48 +/- 0.33, 2.20 +/- 0.44, 2.76 +/- 0.37, 2.15 +/- 0.29 and 2.78 +/- 0.31 mg, respectively. In Alteplase-treated rabbits, only the 3.3 mg/kg dose significantly increased the group P50 by 189% compared to control. Tenecteplase was also effective at increasing the P50 value to 2.21 +/- 0.43 mg if there was a 3-h delay following embolization, but not if there was a 6-h delay before administration. Alteplase was only effective if administered 1 h following embolization where it significantly increased the P50 value to 3.27 +/- 0.40 mg. This study indicates that Tenecteplase has a wide therapeutic range, a therapeutic window of at least 3 h and a durable effect. Moreover, the safety profile for Tenecteplase is similar to that of Alteplase. Tenecteplase does not increase the rate of intracerebral hemorrhage (ICH) above that produced by Alteplase. However, the therapeutic range and window for Alteplase is more limited than that for Tenecteplase. Our preclinical studies suggest that Tenecteplase has a better pharmacological profile than Alteplase and supports further investigation of Tenecteplase in randomized double-blinded clinical trials in stroke patients.     

携带tPA基因转移的真核表达载体的构建研究

目的　构建一种无形成病毒及激活原癌基因之虞的真核表达载体ｐｃＤＮＡ３．１（＋ ）ｔＰＡ，以探讨其对纤溶活性的影响。方法　用分子克隆方法，将人ｔＰＡ ｃＤＮＡ 与真核表达载体ｐｃＤＮＡ３．１（＋ ）重组，并对重组质粒进行ＤＮＡ 测序。结果　重组质粒ｐｃＤＮＡ３．１（＋ ）ｔＰＡ 经Ｋ ｐｎＩ和ＸｂａＩ酶切后，出现了５．４ｋｂ 与 ２．０ｋｂ 两个片段；经ＰｓｔＩ酶切后出现了 ７８ｂｐ，４１４ｂｐ，６２２ｂｐ，２．０Ｋｂ 及４．２Ｋｂ 五个片段；经ＥｃｏＲＩ酶切后，出现了４７２ｂｐ 及６．９Ｋｂ 两个片段；测序结果证明为人全长ｔＰＡ ｃＤＮＡ。结论　该新型表达质粒的构建为探讨基因防治脑梗塞等血栓性疾病提供了新工具，新思路     

High levels of histidine decarboxylase in the striatum of mice and rats

Stachybotrys microspora triprenyl phenol-7 (SMTP-7) is a novel fibrinolytic agent with anti-inflammatory effect. Previous study demonstrated that SMTP-7 further ameliorated infarction volume in a mouse embolic stroke model compared with tissue type plasminogen activator (tPA), but the reason SMTP-7 has more beneficial effect than tPA has not yet been determined. In the present study, we investigated whether SMTP-7 has an intrinsic neuroprotective effect against transient focal cerebral ischemia (tFCI). Sprague-Dawley rats were subjected to tFCI by intraluminal middle cerebral artery occlusion for 2 h. Following induction of tFCI, rats were randomized into two groups based on the agent administered: SMTP-7 group and vehicle group. We examined cerebral infarction volume 24 h after reperfusion, and evaluated superoxide production, the expressions of nitrotyrosine and matrix metalloproteinase-9 (MMP-9), which play major roles in secondary brain injury and hemorrhagic transformation. The findings showed that SMTP-7 significantly suppressed superoxide production, the expression of nitrotyrosine and MMP-9 after tFCI, and consequently attenuated ischemic neuronal damage. These results suggest that SMTP-7 has an intrinsic neuroprotective effect on ischemia/reperfusion injury through the suppression of oxidative stress and MMP-9 activation.