Effects of black cohosh on the plasminogen activator system in vascular smooth muscle cells

Objective

The rhizome of the Cimicifuga racemosa plant (commonly known as black cohosh) has been used for menopausal complaints. Studies regarding the cardiovascular effects of black cohosh are lacking. We investigated the effect of black cohosh on the plasminogen activator system in cultured vascular smooth muscle cells (VSMCs).

Methods

VSMCs were isolated from rat aortae. Expression of plasminogen activator inhibitor type 1 (PAI-1) and tissue-type plasminogen activator (t-PA) proteins were evaluated by Western blot analysis and enzyme-linked immunosorbent assay, respectively. The activities of PAI-1 and t-PA in the conditioned media were assessed by fibrin overlay zymography. A 40% 2-propanol extract of black cohosh was used.

Results

Black cohosh extract (BcEx) stimulated the protein expression of PAI-1, but it did not affect that of t-PA. Vitamin E, a potent antioxidant, inhibited the BcEx-induced increase in PAI-1 expression, while ICI 182,780, an estrogen receptor antagonist, had no effect. Fibrin overlay zymography revealed that BcEx increased the activity of PAI-1 in the conditioned media, while concurrently decreasing that of free t-PA by inducing a binding to PAI-1.

Conclusions

BcEx induces PAI-1 protein expression in the VSMCs likely via an oxidant mechanism. It also stimulates the enzyme activity of PAI-1 and reduces that of free t-PA. These findings suggest that black cohosh might exert a negative influence on fibrinolysis.     

Potentiating effect of endothelial cells on astrocytic plasminogen activator inhibitor type-1 gene expression in an in vitro model of the blood–brain barrier

There is accumulating evidence of the importance of cellular communication between the cells that compose the blood-brain barrier (BBB). Astrocytes are known to affect the expression of tissue-type plasminogen activator (t-PA) and its inhibitor plasminogen activator inhibitor type-1 (PAI-1) in endothelial cells. We investigated the influence of endothelial cells on astrocytic gene expression of PAI-1, protease nexin-1 (PN-1) and t-PA using an in vitro model of the BBB. Primary rat astrocyte-enriched cultures were cocultured with primary adult rat brain microvascular endothelial cells on opposite sides of a transwell membrane. After coculturing for 9–11 days, the cultures were treated with lipopolysaccharide (LPS) for 8 h or 24 h. The levels of PAI-1, PN-1 and t-PA mRNA in untreated and treated monocultures and cocultures were analyzed by Real-Time RT-PCR. Cocultivation of astrocytes and endothelial cells increased astrocytic PAI-1 mRNA expression, and this response was further amplified by LPS treatment. The levels of PN-1 and t-PA mRNA expression in astrocytes were unaffected by cocultivation and/or LPS treatment. Analysis of endothelial PAI-1 and t-PA gene expression revealed increased PAI-1 mRNA levels in cocultured cells, whereas t-PA mRNA levels remained unchanged. These results demonstrate that the cocultivation of astrocytes and endothelial cells induces a pronounced increase in astrocytic PAI-1 gene expression, and that this effect is amplified by LPS treatment. These findings imply an important role for intercellular crosstalk in modulating PAI-1 gene expression within the BBB, under both physiologic and pathophysiologic conditions.     

氧化低密度脂蛋白内皮受体在氧化低密度脂蛋白致内皮细胞损伤中的作用

目的:探讨血凝素样氧化低密度脂蛋白受体(LOX1)在氧化低密度脂蛋白(ox-LDL)致内皮细胞损伤中的作用。方法:采用倒置相差显微镜观察细胞形态的改变;发色底物法检测内皮细胞培养液中的组织纤溶酶原激活物(t-PA)和纤溶酶原激活物抑制剂(PAI-1)的活性;反转录聚合酶链反应(RT-PCR)检测LOX1mRNA表达水平。结果:单纯加入ox-LDL,内皮细胞出现胞体收缩,细胞膜破坏等明显的损伤性改变,培养液中PAI-1活性较对照组高3倍(P<0.05),LOX1mRNA水平表达增高;而当培养液中同时加有LOX1抑制剂polyinosinicacid时,内皮细胞形态损伤不明显,培养液中PAI-1活性低约2.6倍(P<0.05),同时LOX1mRNA水平降低。结论:LOX1介导了ox-LDL对内皮细胞的损伤,可能在血管损伤性疾病的发生中起重要作用。     

同型半胱氨酸诱导血管平滑肌细胞组织因子表达增强

目的: 研究同型半胱氨酸（Hcy）诱导人血管平滑肌细胞（VSMCs）组织因子（TF）表达的作用，及其对核转录因子（NF-κB）活性和诱导型一氧化氮合酶（iNOS）表达的影响。 方法: 培养人脐动脉VSMCs，将不同浓度的Hcy和/或NF-κB抑制剂PDTC与VSMCs孵育不同时间，用RT-PCR方法检测TF mRNA表达，用Western blotting检测核NF-κB水平，用流式细胞术（FCM）检测细胞表面TF蛋白水平和iNOS水平。 结果: 10 μmol/L Hcy作用4 h，TF表达开始升高，500 μmol/L达峰值，Hcy能显著诱导VSMCs表达TF mRNA；对照组VSMCs细胞膜表面有低水平的TF蛋白表达，10 μmol/L Hcy作用4 h即可诱导VSMCs表达TF蛋白，500 μmol/L 达高峰，Hcy能显著诱导VSMCs表达TF；500 μmol/L Hcy作用1 h，细胞核NF-κB水平明显升高，Hcy可诱导VSMCs NF-κB活化;NF-κB抑制剂PDTC可明显抑制Hcy对TF mRNA和细胞TF表达的诱导作用；单独Hcy作用不能诱导VSMCs表达iNOS。 结论: Hcy能显著诱导VSMCs表达TF，增强VSMCs的NF-κB活化，从而介导TF基因表达和蛋白合成，通过NF-κB介导VSMCs表达TF可能是Hcy导致AS、血栓形成的重要机制。     

Tissue plasminogen activator, plasminogen activator inhibitors, and activator-inhibitor complex in liver disease.

AIMS--To identify the relative contribution of plasminogen activators, particularly tissue plasminogen activator (t-PA) and specific plasminogen activator inhibitors (PAI-1, PAI-2), to the fibrinolytic changes associated with various types of liver disease or severe chemical and physical damage to the liver. METHODS--Platelet rich (PRP) and platelet poor plasma (PFP) from patients with alcoholic cirrhosis, primary biliary cirrhosis, hepatic malignancy, or paracetamol overdose, or who were undergoing partial hepatectomy or liver transplantation, were assayed for t-PA, PAI-1, t-PA-PAI-1 complex and PAI-2 antigen values using specific enzyme linked immunosorbent assays (ELISAs) developed in this laboratory. RESULTS--Appreciable increases in the plasma concentration of t-PA, PAI-1, and t-PA-PAI-1 were seen in patients with alcoholic cirrhosis, primary biliary cirrhosis, and hepatic malignancy. Liver damage due to paracetamol overdose and partial hepatectomy both resulted in a striking increase in plasma PAI-1 concentration, although concentrations of t-PA and t-PA-PAI-1 complex were less affected. Concentrations of t-PA, PAI-1, and t-PA-PAI-1 complex returned to near normal values after successful liver transplantation in a patient with chronic active hepatitis. PAI-2 was also detected in several patients with chronic liver disorders. CONCLUSIONS--Haemorrhage due to fibrinolytic bleeding is commonly associated with liver disease. The patients studied here all had appreciable increases in circulating t-PA antigen concentrations. This was associated with increased concentrations of PAI-1 antigen and t-PA-PAI-1 complex and the balance between activator and inhibitor did not result in systemic plasmin generation. Reduced PAI-1 activity in cirrhosis or a critical difference in the ratio of t-PA to PAI-1 concentrations may explain the enhanced plasminogen activator activity previously noted in cirrhosis but not metastatic disease. Reduced hepatic clearance of t-PA and t-PA-PAI-1 complex due to impaired liver function may account for increased concentrations of free and complexed t-PA.     

Increase of tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor type 1 (PAI-1) in plasma after thrombolytic therapy of patients with myocardial infarction. A randomised,placebo-controlled study

Based on recent studies we hypothesised that the marked generation of thrombin in patients who undergo coronary thrombolysis was associated with substantial deviations of endogenous tissue-type plasminogen activator (t-PA) and the plasminogen activator inhibitor type 1 (PAI-1) in plasma.In the present placebo-controlled study we observe in 20 patients treated with recombinant t-PA (rt-PA) a marked increase (p<0.001) of antigen concentrations in plasma of endogenous t-PA and PAI-1 during the first 12h after initiation of treatment. The concentrations of endogenous t-PA and PAI-1 in plasma correlated significantly (p<0.05) with an estimate of generated thrombin in vivo, determined as plasma concentrations of thrombin-antithrombin III complexes. We conclude that the generation of coagulant activity following coronary thrombolysis is associated with an increased synthesis and/or release of endogenous t-PA and PAI-1, which suggests that the procoagulant condition involves an altered functional state of the vascular endothelium.     

抗PAI抑制作用的组织纤溶酶原激活剂在大肠杆菌中的表达

目的:构建t-PA活性不被PAI-1抑制的新一代t-PA突变体。方法:根据t-PA的结构特点,去除t-PA分子中的指状区、表皮生长因子和Kringle1区,以含全长t-PA编码区序列的pUC18质粒为模板,经PCR扩增编码氨基酸1~3和176~527位的截短式t-PADNA序列;并将该t-PA分子中的PAI-1结合位点,即第373~384位核苷酸(AAGCACAGGAGG)突变为(GCGGCCGCGGCG),相应的氨基酸KHRR则变为AAAA。结果:测序证实,t-PA突变体的DNA序列正确,将其克隆于大肠杆菌表达载体中,并在大肠杆菌中得到高效表达。表达蛋白占总菌体蛋白的30%,以包涵体形式存在。经蛋白质变性、复性,得到有活性的t-PA突变体。t-PA突变体与PAI-1反应后t-PA的活性未受到抑制。结论:t-PA突变体可能是一种用于治疗心肌梗死和脑血栓等血栓性疾病的强效且剂量要求低的新型生物工程药物。     

辛伐他汀对尼古丁诱导脐静脉 内皮细胞分泌t-PA和PAI-1的影响

目的: 研究不同浓度辛伐他汀对尼古丁诱导人脐静脉内皮细胞(HUVECs)分泌组织纤溶酶原激活物(t-PA)和1型纤溶酶原激活物抑制剂(PAI-1)及其基因表达的影响。方法: 将3-6代体外培养的HUVECs随机分为对照组、尼古丁组及不同浓度辛伐他汀组,辛伐他汀组分别以1、10、100 μmol/L辛伐他汀预处理细胞2 h,再以100 μmol/L尼古丁孵育24 h。酶联免疫吸附双抗体夹心法(ELISA)检测细胞上清液t-PA和PAI-1含量;逆转录聚合酶链反应(RT-PCR)检测细胞t-PA和PAI-1 mRNA的表达。结果: 尼古丁组PAI-1分泌和mRNA表达较对照组显著升高(P<0.05)。不同浓度辛伐他汀组PAI-1分泌和mRNA表达均较尼古丁组显著降低,且PAI-1分泌和mRNA表达的降低呈浓度依赖性(均P<0.05),以100 μmol/L辛伐他汀组最为显著。100 μmol/L辛伐他汀组PAI-1分泌和mRNA表达与对照组比较,无显著差异(P>0.05)。尼古丁组t-PA mRNA表达较对照组显著降低(P<0.05)。10、100 μmol/L辛伐他汀组t-PA mRNA表达较尼古丁组显著升高(P<0.05),各组间t-PA分泌无显著差异(均P>0.05)。结论: 在体外,辛伐他汀可降低尼古丁所致的PAI-1分泌和mRNA的表达,并升高t-PA mRNA的表达,从而逆转尼古丁介导的HUVECs纤溶活性减低。     

The relationship between plasma t-PA and PAI-1 levels is dependent on epistatic effects of the ACE I/D and PAI-1 4G/5G polymorphisms

Thrombus formation and degradation is partly due to a complex interplay between tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor 1 (PAI-1). There is accumulating evidence that plasma levels of t-PA and PAI-1 may be influenced by an interaction between the fibrinolytic and renin-angiotensin systems. The goal of this study was to conduct an exploratory data analysis to determine whether there is evidence that the relationship (i.e. correlation) between plasma t-PA and PAI-1 is influenced by interactive effects of the angiotensin converting enzyme (ACE) insertion/deletion (I/D) and plasminogen activator inhibitor 1 (PAI-1) 4G/5G polymorphisms in a sample of 50 unrelated African Americans and 117 unrelated Caucasians. In a single-locus analysis, no evidence for heterogeneity of plasma t-PA and PAI-1 correlations among either ACE I/D or PAI-1 4G/5G genotypes was detected. However, using the combinatorial partitioning method for exploratory data analysis, we identified evidence that is suggestive of heterogeneity of plasma t-PA and PAI-1 correlations among multilocus ACE I/D and PAI-1 4G/5G genotypes in African American females, Caucasian females, Caucasian males, but not African American males. From these results, we propose as a working hypothesis that the correlation between plasma t-PA and PAI-1 may be dependent on epistatic effects of the ACE I/D and PAI-1 4G/5G polymorphisms. This study supports the idea that interactions between the fibrinolytic and renin-angiotensin systems play an important role in the genetic architecture of plasma t-PA and PAI-1.     

Quantification of a specific inhibitor (PAI-1) of tissue-type plasminogen activator (t-PA) in the plasma

In human plasma, the activation of plasminogen by tissue plasminogen activator (t-PA) is a fibrin localized process which allows the specific dissolution of thrombi. Most of the t-PA circulates as a complex with its inhibitor, PAI-1, which thereby regulates its activity. In the present work the authors have studied the kinetics of inhibition of t-PA by PAI-1 and have developed an assay for its specific detection. The assay is performed in microtitration plates containing a solid-phase fibrin network, as follows: the source of inhibitor is mixed with solutions containing increasing amounts of t-PA, then the residual t-PA is separated by means of a solid-phase fibrin support and detected with a coupled reaction using a plasmin selective chromogenic substrate. The change in absorbance is measured in a microtiter plate reader and converted to t-PA activity by reference to a standard curve. The residual t-PA activity is inversely proportional to the concentration of PAI-1. The quantitation of PAI-1 is based on the variation of the dissociation constant of the fibrin/t-PA interaction obtained in the presence of the inhibitor. Since other serine-protease inhibitors do not interfere with the assay, the method is specific for PAI-1 and can be safely used in other biological fluids.     

慢性肾脏疾病血清和尿液纤溶活性物质的改变及临床意义

目的探讨慢性肾脏疾病血清和尿液纤溶活性物质的改变及其临床意义。方法选择38例慢性肾小球肾炎(CGN),28例肾病综合征(NS),36例非透析治疗的慢性肾功能不全(CRF)和20例正常对照作为研究对象,应用ELISA法检测血清和尿液中组织型纤溶酶原激活剂(t-PA)和纤溶酶原激活物抑制剂-1(PAI-1)的浓度,同时分析尿中t-PA和PAI-1的水平与血t-PA、PAI-1、血肌酐和24h尿蛋白总量之间相关性。结果慢性肾脏疾病出现血清t-PA、PAI-1升高,尿液t-PA、PAI-1降低,其中尿液t-PA、PAI-1的改变独立于血清,不受血肌酐和24h尿蛋白定量的影响。结论慢性肾脏疾病患者存在纤溶活性物质的异常,其中尿液纤溶活性物质的改变可反应肾脏内皮细胞损伤。     

Neutralization of plasminogen activator inhibitor-1 (PAI-1) by activated protein C is species-dependent

The interaction of bovine and human activated protein C (APC) with type-1 plasminogen activator inhibitor (PAI-1) was studied in a cell-free system. Human plasma and a preparation of PAI-1 obtained from human endothelial cell cultures were used as sources of PAI-1. Bovine APC was able to neutralize PAI-1 inhibitory activity present in both sources in a dose-dependent manner; the concentration of bovine APC required to produce 50% (C₅₀) neutralization of endothelial PAI-1 (0.5nM) was 4 μg/ml (64nM). Moreover, when complexes between tissue plasminogen activator (t-PA, 60ng/ml, 0.84nM) and PAI-1(18.8ng/ml, 0.35nM) were incubated with bovine APC, the amount of such complexes decreased as a function of the concentration of added APC (C₅₀ = 2 μ/ml, 32 nM), and a concomitant increase in the amount of residual t-PA activity was observed. This effect was due to the formation of APC⊎PAI-1 complexes as detected by immunoblotting with monoclonal antibodies directed against PAI-1. By this mechanism bovine APC prevented the initial reaction between PAI-1 and t-PA and interfered with the stability of complexes between t-PA and PAI-1. The latter observation suggests that complexes between t-PA and PAI-1 may dissociate in the presence of bovine APC. In contrast with these findings, when the experiments were performed in an entirely human homologous cell-free system, human APC did not form a complex with PAI-1 and no effect either on PAI-1 or on the stability of preformed t-PA ⊎ PAI-1 complexes was observed. The results indicate that the neutralization of PAI-1 by APC is a phenomenon induced by interspecies molecular interactions.     

Chronic glucocorticosteroid administration modulates the response of the fibrinolytic system to bacterial lipopolysaccharide

An in vivo rat model was used to study the effect of chronic glucocorticosteroid administration on the response of the fibrinolytic system to lipopolysaccharide (LPS). Male Lewis rats were injected subcutaneously for 30 consecutive days with either saline or saline containing 0.1 μg/g dexamethasone. On the thirty-first day, the rats were challenged with intraperitoneal injections of LPS (0.5 μg/g). Plasma and selected tissues (liver, kidney, heart, lung, muscle, and fat) were removed for analysis at various times after LPS injection. LPS treatment caused a rapid rise in plasma type 1 plasminogen activator inhibitor (PAW) activity in both groups. However, in the dexamethasone-pretreated animals a transient decline in PAI-1 activity was detected at 4 h, coinciding with a transient increase in plasma tissue plasminogen activator (t-PA) activity and a marked elevation of t-PA messenger RNA (mRNA) levels in the lung. Plasma t-PA activity returned below basal levels by 8 h and did not differ between the two groups at later times. At 24h, PAI-1 activity remained significantly elevated in the dexamethasone-pretreated rats while declining in the saline-pretreated rats. A greater induction of PAI-1 mRNA was evident in the dexamethasone-pretreated rats in all six tissues examined. Notably, a 3-fold greater increase in PAI-1 mRNA was detected in the liver at 24h, corresponding with the sustained elevation in plasma PAI-1 activity at this time. Collectively, these data suggest that chronic glucocorticosteroid treatment potentiates the induction of both t-PA and PAI-1 gene expression by LPS, resulting in an initial transient increase in plasma t-PA activity followed by a more sustained elevation in plasma PAI-1 activity.     

卡托普利对家兔急性心肌梗塞早期血小板活化及纤溶活性的影响

本研究观察了家兔急性心肌梗塞（ＡＭＩ）早期血浆血小板α－颗粒膜蛋白（ＧＭＰ－１４０）、血栓素Ｂ２（ＴＸＢ２）、组织型纤溶酶酶原激活剂（Ｔ－ＰＡ）及其抑制物（ＰＡＩ－１）水平的变化及卡托利干预的ＰＡ活性显著降低；相关分析表明，血浆ＧＭＰ－１４０浓度与ＰＡＩ－１活性显著正相关。卡托普利干预后，血浆ＧＭＰ－１４０浓度、ＴＹＸＢ２浓度、Ｔ－ＰＡ浓度、ｔ－ＰＡ含量、ＰＡＩ－１活性均明显降低，而ｔ－ＰＡ活性显     

Presence of a fast-acting specific inhibitor of plasminogen activator in human parotid saliva

The plasminogen activator in parotid saliva has recently been characterized as a tissue-type plasminogen activator (t-PA). In this study stimulated parotid saliva from 21 healthy volunteers was analysed for (a) t-PA activity using the fibrin plate assay or a bioimmunoassay coupled to the plasmin-specific chromogenic substrate S-2251, (b) t-PA antigen using an enzyme-linked immunospecific assay and (c) activity of the specific fast-acting plasminogen activator inhibitor (t-PA inhibitor) assayed by its inhibitory effect on one-chain t-PA added to the samples. In parotid saliva the median t-PA activity was 0.2 IU ml-1 (normal range 0.05-0.35 IU ml-1), but activity of free t-PA could not be demonstrated by bioimmunoassay in any sample. The mean antigen concentration of t-PA was 2.2 IU ml-1 in the 10 samples with levels above the detection limit (0.5 IU ml-1). High activity of t-PA inhibitor was demonstrated in all parotid salivas, and the mean inhibitory effect on t-PA was 13.2 IU t-PA quenched per millilitre. This study thus demonstrates a fibrinolytic system in parotid saliva characterized by high t-PA inhibitor activity and relatively low concentration of inactive, probably complex-bound, t-PA which is activated in the presence of fibrin.     

Levels of tissue-type plasminogen activator and plasminogen activator inhibitor 1 in disseminated intravascular coagulation syndrome

Since disseminated intravascular coagulation (DIC) may directly reflect the abnormal regulation of the fibrinolytic system by endothelial cells, we have measured the levels of tissue-type plasminogen activator (t-PA), type 1 PA inhibitor (PAI-1) and t-PA . PAI-1 complex which is formed as a result of interaction on the two factors, in the plasma of patients with DIC (n = 51) and healthy controls (n = 42). Antigens of t-PA, PAI-1 and t-PA . PAI-1 complex were significantly increased in the DIC plasma (36.4 +/- 25.1, 106.8 +/- 54.7 and 46.6 +/- 34.5 ng/ml, respectively) compared with those in normal plasma (8.5 +/- 4.3, 54.4 +/- 21.2 and 8.6 +/- 3.5 ng/ml, respectively). The molar ratio of t-PA to PAI-1 was much higher in the DIC plasma (1:3) than in normal plasma (1:6), which caused enhancement of the whole fibrinolytic activity in the DIC plasma. These changes resulted in significant consumption of plasminogen, alpha 2-plasmin inhibitor (alpha 2-PI) and a significant increase of plasmin . alpha 2-PI complex (PPI) and D-dimer. These results suggest that t-PA and its specific inhibitor PAI-1 both of which are secreted from endothelial cells into blood, play an important role on the progress of DIC.     

CCK-8抑制LPS作用下大鼠肺间质巨噬细胞NF-κB活性的cAMP-PKA通路研究

目的： 用cAMP激动剂forskolin和PKA抑制剂H-89，探讨八肽胆囊收缩素（CCK-8）抑制LPS作用下大鼠肺间质巨噬细胞（PIMs）核因子-κB （NF-κB）活性的cAMP-PKA信号通路机制。 方法： 分离纯化大鼠PIMs。用电泳迁移率改变分析（EMSA）法检测大鼠PIMs中NF-κB活性，用Western blotting分析IκB-α蛋白水平。 结果： 正常对照组大鼠PIMs核内未检测到与特异性寡核苷酸探针相结合的NF-κB，LPS组细胞内NF-κB活性明显高于正常对照组（P<0.01），胞浆中IκB-α水平显著低于正常对照组（P<0.01）。CCK组和Fsk组细胞内NF-κB活性和胞浆中IκB-α含量均无明显差异（P>0.05）。CCK+LPS组和Fsk+LPS组，细胞内NF-κB活性均低于LPS组（P<0.05），IκB-α含量均高于LPS组（P<0.01）。LPS+CCK+H-89组NF-κB活性高于CCK+LPS组（P<0.01），而IκB-α蛋白水平低于CCK+LPS组（P<0.01）。 结论： cAMP-PKA信号通路的活化可抑制LPS诱导的大鼠PIMs细胞内NF-κB活性升高和IκB-α蛋白水平的降低，CCK-8的抗炎作用是通过激活cAMP-PKA信号通路进而抑制NF-κB活性来实现的。