Immunoelectron Microscopic Localization of Plasminogen Activator Inhibitor Type 1 (PAI-1) in Smooth Muscle Cells from Morphologically Normal and Atherosclerotic Human Arteries

Vascular wall fibrinolytic system proteins are believed to play a pivotal role in atherogenesis. Tissue-type plasminogen activator (t-PA) and urokinase plasminogen activator (u-PA) influence persistence of luminal thrombi and proteolysis of extracellular matrix, respectively. The major physiologic inhibitor of t-PA and u-PA is plasminogen activator inhibitor type 1 (PAI-1). All three of these fibrinolytic system proteins have been detected in vascular endothelial cells, smooth muscle cells, and macrophages by light microscopic immunohistochemistry. This study was undertaken to delineate, by immunoelectron microscopy, the loci of PAI-1 in smooth muscle cells from intact morphologically normal and atherosclerotic human arteries as well as in isolated and cultured smooth muscle cells from arteries. In intact vessels, PAI-1 immunoreactivity was associated with contractile filaments in cells in both normal and atherosclerotic tissues. Lipid-laden smooth muscle cells in atherosclerotic vessels were mainly of the synthetic phenotype and displayed lesser amounts of PAI-1 associated with rough endoplasmic reticulum and contractile filaments. Isolated smooth muscle cells exhibited either a contractile or synthetic phenotype. In the cells with a contractile phenotype, PAI-1 was associated with the contractile elements, whereas in the cells with a synthetic phenotype, the PAI-1 was associated predominantly with elements of the endoplasmic reticulum. Because PAI-1 is associated predominantly with contractile filaments in smooth muscle cells, the net amount of immunodetectable PAI-1 appears to be greater in contractile compared with synthetic phenotype cells.     

Immunoelectron Microscopic Localization of Plasminogen Activator Inhibitor Type 1 (PAI-1) in Smooth Muscle Cells from Morphologically Normal and Atherosclerotic Human Arteries

Vascular wall fibrinolytic system proteins are believed to play a pivotal role in atherogenesis. Tissue-type plasminogen activator (t-PA) and urokinase plasminogen activator (u-PA) influence persistence of luminal thrombi and proteolysis of extracellular matrix, respectively. The major physiologic inhibitor of t-PA and u-PA is plasminogen activator inhibitor type 1 (PAI-1). All three of these fibrinolytic system proteins have been detected in vascular endothelial cells, smooth muscle cells, and macrophages by light microscopic immunohistochemistry. This study was undertaken to delineate, by immunoelectron microscopy, the loci of PAI-1 in smooth muscle cells from intact morphologically normal and atherosclerotic human arteries as well as in isolated and cultured smooth muscle cells from arteries. In intact vessels, PAI-1 immunoreactivity was associated with contractile filaments in cells in both normal and atherosclerotic tissues. Lipid-laden smooth muscle cells in atherosclerotic vessels were mainly of the synthetic phenotype and displayed lesser amounts of PAI-1 associated with rough endoplasmic reticulum and contractile filaments. Isolated smooth muscle cells exhibited either a contractile or synthetic phenotype. In the cells with a contractile phenotype, PAI-1 was associated with the contractile elements, whereas in the cells with a synthetic phenotype, the PAI-1 was associated predominantly with elements of the endoplasmic reticulum. Because PAI-1 is associated predominantly with contractile filaments in smooth muscle cells, the net amount of immunodetectable PAI-1 appears to be greater in contractile compared with synthetic phenotype cells.     

Hyperthermia stimulates plasminogen activator inhibitor type 1 expression in human umbilical vein endothelial cells in vitro.

The effect of exposure to hyperthermia on the fibrinolytic potential of human umbilical vein endothelial cells (HUVEC) in culture was studied. HUVEC responded to exposure to 42 degrees C with a time-dependent increase in plasminogen activator inhibitor type 1 (PAI-1) activity and antigen accompanied by a four- to fivefold increase in PAI-1 specific m-RNA and a decrease in tissue-type plasminogen activator (t-PA) antigen. The effect of 8 hours exposure to hyperthermia on PAI-1 activity and antigen could not be reversed by reexposure of the cells to 37 degrees C for 24 hours as evidenced by continuously increased amounts of PAI-1 released into the conditioned media. t-PA release, however, decreased during the 24-hour period at 37 degrees C after exposure to hyperthermia. No difference in PAI-1 antigen present in the extracellular matrix of heat treated HUVEC as compared to HUVEC kept at 37 degrees C could be found. Our data supports the idea that hyperthermia is one stress factor that influences the fibrinolytic potential of endothelial cells.     

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。     

Extracellular and cell-associated localizations of plasminogen activators and plasminogen activator inhibitor-1 in cultured endothelium

The extracellular localizations of urokinase-type plasminogen activator (uPA), tissue-type plasminogen activator (tPA), and plasminogen activator inhibitor-1 (PAI-1) were examined in cultured bovine capillary endothelial cells (BCEs) by an immunofluorescence method using BCEs treated with or without saponin and focal contact preparations. The specific immunofluorescence of cell surface uPA showed a patchy or strand-like distribution and was colocalized with vinculin strands indicating that uPA secreted from BCEs was mainly deposited at the cell surface of focal contacts. BCEs at a subconfluent density showed a higher intensity of specific immunofluorescence for uPA than when they were at a confluent density. tPA was observed over the dorsal surface of cultured BCEs and accentuated at their margins, suggesting that tPA was diffusely distributed on the luminal surface of BCEs in vivo. PAI-1 was distributed in the extracellular matrix under cultured BCEs. These findings suggest that uPA and PAI-1 are located under BCEs participating in the regulation of proteolytic activities provoked by plasminogen-PAs-plasmin system in vivo. The localization of tPA appears to be consistent with its function, which is to maintain the fluidity of the blood and to initiate thrombolysis in vivo.     

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.     

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.     

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。     

Plasminogen activators are involved in keratinocyte and fibroblast migration in wounded cultures in vitro

During skin repair both keratinocytes and fibroblasts migrate into the wounded area. In the process of cell migration, controlled proteolytic degradation of the extracellular matrix occurs. It has been suggested that the plasminogen activator system is involved in such proteolytic processes occurring during wound healing.The role of plasmin, urokinase-type plasminogen activator (u-PA) and tissue-type plasminogen activator (t-PA) in keratinocyte and fibroblast migration in vitro was examined in the present study. Confluent cultures of normal human keratinocytes, human squamous cell carcinoma (SCC-4), SV40 transformed keratinocytes (SVK-14) and human fibroblasts were mechanically wounded and the repopulation of the denuded area was examined. Migration of cells into the denuded area could be inhibited by neutralizing antibodies against t-PA or u-PA, or the serine protease inhibitor Trasylol in all cell types studied. For detailed study on involvement of u-PA and t-PA in migration processes wounded cultures of SVK14 cells were used. Both t-PA and u-PA activity could be detected at the migrating edge of SVK14 cultures as revealed by zymography and by immunocytochemistry using polyclonal antibodies.Our results demonstrate the direct involvement of not only u-PA but also t-PA in migration of keratinocytes and fibroblasts in wounded cultures in vitro.     

尼古丁对血管内皮细胞释放t-PA及PAI-1的影响

目的: 研究尼古丁对人脐静脉内皮细胞(HUVECs)释放组织型纤溶酶原激活物(t-PA)和纤溶酶原激活物抑制物-1(PAI-1)的影响。方法: HUVECs培养后接种于24孔培养板中,随机分为对照组及实验组,分别进行以下实验。(1)以0.1、1、10、100 μmol/L 尼古丁孵育HUVECs,12 h后收集各组上清液;(2)以100 μmol/L尼古丁与HUVECs孵育0、4、6、8、12 及24 h,收集各组上清液。采用ELISA法测定各组t-PA和PAI-1的浓度。结果: HUVECs与不同浓度尼古丁孵育12 h后,100 μmol/L尼古丁组PAI-1蛋白较对照组明显增加(P<0.01);0.1、1及10 μmol/L尼古丁组PAI-1蛋白与对照组比较,均无显著差异(均P>0.05);各浓度组t-PA蛋白与对照组比较,均无显著差异(均P>0.05)。HUVECs 与100 μmol/L的尼古丁分别孵育4 、6 、8 、12 及24 h,各组PAI-1蛋白均较对照组明显升高(P<0.05),且其升高呈时间依赖性;各组t-PA与对照组比较,均无显著差异(均P>0.05)。结论: 尼古丁可抑制HUVECs的纤溶活性,对内皮细胞具有损伤作用。     

Promigratory Effect of Plasminogen Activator Inhibitor-1 on Invasive Breast Cancer Cell Populations

The urokinase-type plasminogen activator (uPA) system is a dynamic complex in which the membrane receptor uPAR binds uPA that binds the plasminogen activator inhibitor (PAI)-1 localized in the extracellular matrix, resulting in endocytosis of the whole complex by the low-density lipoprotein receptor-related protein (LRP). High expression of PAI-1 is paradoxically associated with marked tumor spreading and poor prognosis. We previously reported a nonproteolytic role of the [uPAR:uPA:PAI-1:LRP] complex operative in cell migration. Here we explored whether matrix PAI-1 could be used as a migration support by human breast cancer cells. We showed that the uPA system and LRP are localized at filopodia of invasive cells, and that formation/internalization of the [uPAR:uPA:PAI-1:LRP] complex is required for attachment and migration of cancer cells on plastic and on a PAI-1 coat. PAI-1 increased both filopodia formation and migration of cancer cells suggesting a chemokine-like activity. Migration velocity, expression of the uPA system, use of the [uPAR:uPA:PAI-1:LRP] complex to migrate, and promigratory effects of PAI-1 paralleled cancer cell invasiveness. Phenotyping and functional analysis of invasive cancer cell subclones indicated that different cell subpopulations may use different strategies to migrate depending on both the environment and their expression of the uPA system, some of them taking advantage of abundant available PAI-1.     

Induction of t-PA Synthesis with intravenous bolus injection of vitamin A palmitate in vitamin a deficient rats

Retinoic acid and vitamin A palmitate induce tissue-type plasminogen activator (t-PA) synthesis in cultured human umbilical vein endothelial cells (HUVEC) in vitro without alteration of plasminogen activator inhibitor-] (PAI-1) synthesis. Therefore the effect of intravenous bolus injection of water-miscible vitamin A palmitate on the blood fibrinolytic system was investigated in normal and vitamin A deficient rats. In 5 normal rats, injection of 200000 IU/kg of vitamin A palmitate did not induce a significant increase in euglobulin fibrinolytic activity, t-PA antigen and PAI activity in plasma nor of t-PA and PAI-1 mRNA in the heart within 240 min after injection. In groups of 3–6 vitamin A deficient rats, injection of 200000IU/kg of vitamin A palmitate induced a significant increase in t-PA antigen in plasma (1.9-fold after 60 min and 2.9-fold after 120 min), but no significant increase in plasma euglobulin fibrinolytic activity. A 2-fold increase in PAI activity was observed 90 min after injection of both vitamin A palmitate as well as of its solvent, suggesting that this induction was non-specific. The increase in t-PA antigen coincided with a significant increase in t-PA mRNA in heart tissue: 2.3-fold after 60 min and 4.3-fold after 120 min. PAI-1 mRNA in heart tissue increased 3.6-fold 90 min after injection with vitamin A palmitate but, surprisingly, not after injection of solvent.It is concluded that intravenous bolus injection of vitamin A palmitate induces a specific increase of t-PA mRNA in the heart and of t-PA antigen secretion in plasma of vitamin A deficient rats.     

Vascular endothelial growth factor, epidermal growth factor and fibroblast growth factor-4 and -10 stimulate trophoblast plasminogen activator system and metalloproteinase-9

Trophoblast invasion, accompanied by degradation of extracellular matrix, is crucial to normal pregnancy development, whereas shallow placental invasion and implantation likely plays a role in the subsequent development of pre-eclampsia. The growth factors vascular endothelial growth factor (VEGF), epidermal growth factor (EGF) and fibroblast growth factor (FGF) are placental growth factors that activate degradation of extracellular matrix. We determined the effect of VEGF, EGF, FGF-2, FGF-4 and FGF-10 on the plasminogen activator system of first trimester cytotrophoblasts cultured in vitro. We studied the activity of urokinase plasminogen activator (uPA), its inhibitor plasminogen activator inhibitor-1 (PAI-1), and 92 kDa gelatinase-B (matrix metalloproteinase-9, MMP-9), using protein gel and reversed gel zymography. The expression pattern of FGF-4 and FGF-10 in human placental sections was determined by immunohistochemistry. FGF-4 was expressed in first trimester villi stroma, primarily in endothelial cells. FGF-10 expression was localized to first trimester extravillous trophoblasts. VEGF, EGF, FGF-4 and FGF-10, but not FGF-2, stimulate the activity of trophoblast uPA, PAI-1 and MMP-9. These results support the hypothesis that specific growth factors modulate the invasive potential of trophoblasts, and therefore may play an important role in early placental development. Our findings may contribute to the understanding of the pathophysiology of diseases associated with shallow placentation, such as pre-eclampsia.     

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.     

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。