Hormonal and immunological characterization of the cell-associated plasminogen activators produced by cultured rat granulosa cells

The hormonal induction and immunological reactivities of the cell-associated plasminogen activators (PAs) produced by granulosa cells obtained from the ovaries of diethylstilbestrol-implanted immature rats were studied. FSH and other cAMP-inducing ligands, including cholera toxin, forskolin, and 8-bromo-cAMP, elevated the PA activity of granulosa cells in a concentration-dependent manner during a 4-h culture, with an approximate 10-fold maximal increase in PA activity compared to control cells. Negligible levels of PA activity were observed in the extracellular medium in the absence or presence of hormones. The PA induced by FSH or cAMP in intact cells was progressively neutralized during the 4-h culture by increasing amounts of antibodies to the tissue-type PA (tPA), but not by an IgG fraction against the urokinase-type PA (UK-PA). However, solubilization of granulosa cells with Triton X-100 revealed the presence of intracellular UK-PA activity in both FSH-treated and control cells that consisted of about 20% of the total cellular PA activity. Electrophoretic analysis of extracts from solubilized granulosa cells indicated the presence of three peaks of PA activity. A PA with a Mr of 70,000 was induced by FSH, was completely inactivated by tPA antibodies, and required fibrin for full activity. A 40,000 Mr fibrin-independent PA was also stimulated by FSH and was partially inhibited by UK-PA antibodies, but not by anti-tPA immunoglobulin G. A third peak of PA activity comigrated with a human UK-PA standard at a Mr of 33,000, was fully neutralized by UK-PA antibodies, and was largely present only in control cells. These results suggest that during the first hours of granulosa cell development, FSH via cAMP induces the production of a cell-surface tPA, while both FSH-treated and control cells synthesize intracellular UK-PAs. Hormonal regulation of the production and activities of these cellular enzymes may allow the expression of specific differentiated functions of developing granulosa cells.     

Regulation of the synthesis and secretion of plasminogen activators by endothelial cells

Plasminogen activators (PAs) play a role in fibrinolysis, tissue remodelling, tumor invasion, and reparative processes. Vascular endothelial cells produce tissue-type PA (t-PA), an important regulator of fibrinolysis, and PA inhibitor 1. They can also synthesize a second type of PA, urokinase-type PA (u-PA). The regulation of synthesis and secretion of these PAs by human and bovine endothelial cells in vitro is reviewed. The synthesis of t-PA and u-PA varies between endothelial cells, depending on their vascular origin. The production of PA activity by endothelial cells is regulated at various levels: (1) induction of newly synthesized t-PA or u-PA molecules; (2) rapid release of t-PA from endothelial cells; (3) conversion of single-chain u-PA in the more active two-chain u-PA; (4) interaction of PAs with cellular receptors and matrix components, and (5) interaction of PAs with specific inhibitors.     

Interactions between cultured bovine arterial endothelial and smooth muscle cells; studies on the release of prostacyclin by endothelial cells.

The release of prostacyclin by endothelial cells (EC) in culture was studied after exposure to two toxic stimuli (UV light or dimethylsulfoxide-soluble smoke particles (DSP)) or to medium conditioned by smooth muscle cells (SMC), basically or after injury to the SMC. An activity stimulating the release of prostacyclin was found together with growth inhibiting activity from arterial SMC, but dissociated from growth stimulating activity. The prostacyclin stimulating activity was increased when SMC were exposed to UV light, while DSP caused a decrease. EC directly exposed to UV light or DSP generally released more prostacyclin than controls. One exception was very low concentrations of DSP. UV light induced a burst of release in contrast to DSP where a continuous release after a two hours lag period was seen. It is concluded that EC will increase the release of prostacyclin in response to injury but the release pattern will depend on the kind and doses of the stimulus. SMC release prostacyclin stimulating activity for EC, which can be modified by exposure to toxic stimuli. The results might have applications for atherogenesis.     

Effect of various plasminogen activators on prostacyclin synthesis in cultured vascular cells

In this study, we examined the effects of various plasminogen activators on arachidonic acid release and prostacyclin biosynthesis in cultured rat aortic smooth muscle cells and bovine pulmonary artery endothelial cells. Prostacyclin was the major product formed from arachidonic acid in aortic smooth muscle cells and endothelial cells. When intact cells were incubated with streptokinase, one of the plasminogen activators, a significant stimulatory effect on prostacyclin biosynthetic activity in cells was evident without any cellular damage at all concentrations used (1-5,000 units/ml). Streptokinase also caused a marked release of arachidonic acid. However, when it was incubated with cell-free homogenates and [3H]arachidonic acid, it did not show any effects on prostacyclin biosynthesis. The addition of urokinase and tissue-type plasminogen activator had no effect on prostacyclin biosynthesis. Urokinase stimulated the release of arachidonic acid from cells, but it did not show any effect on prostacyclin release at any concentration of urokinase (1-5,000 units/ml). The release of arachidonic acid and the increased prostacyclin synthesis were not observed when tissue-type plasminogen activator was added. These results indicate that, among various plasminogen activators investigated, only streptokinase causes the release of arachidonic acid and prostacyclin. This could be a beneficial effect in thrombolytic therapy.     

Differences between binding of one-chain and two-chain tissue plasminogen activators to non-cross-linked and cross-linked fibrin clots

Interaction of tissue plasminogen activator (t-PA) with fibrin plays a key role in regulation of plasminogen activation and clot dissolution. Previous investigations of t-PA-fibrin interaction, using incorporation of t-PA into polymerizing fibrin clots, have suggested that no significant differences exist in the binding of one-chain or two-chain t-PA to non-cross-linked or cross-linked fibrin. In the present study, binding of 125I-labeled and affinity-purified one-chain and two-chain forms of t-PA to preformed non-cross-linked or cross-linked, sonicated suspension of fibrin was investigated. Interaction of one-chain t-PA with cross-linked fibrin involved a single type of binding site with dissociation constant (kd) of 0.58 mumol/L and a stoichiometry (n) of 1.5. Interaction of one-chain t-PA with non-cross-linked fibrin, however, involved two classes of binding sites with dissociation constants of 0.32 and 1.5 mumol/L and corresponding number of binding sites equal to 0.57 and 2.0, respectively. In contrast to the binding of one-chain t-PA to cross-linked fibrin by a limited number of sites, two-chain t-PA appeared to involve a considerably greater number of sites (minimum six) whose dissociation constant was 3.2 mumol/L. Interaction of two-chain t-PA with non-cross-linked fibrin also showed the presence of many binding sites (minimum seven) with approximate dissociation constant of 6.4 mumol/L, as well as a few (n = 0.012) high-affinity sites with a kd of 0.011 mumol/L epsilon-Aminocaproic acid did not completely reverse the binding of either one-chain t-PA or two-chain t-PA to fibrin. The present findings suggest that the fibrin-binding properties of t-PA undergo considerable changes on proteolytic conversion from one-chain to two-chain t-PA, catalyzed under physiologic conditions by plasmin. The cleavage of one-chain t-PA to two-chain t-PA allows to bind to a large number of low-affinity binding sites on fibrin. Cross-linking of fibrin by factor XIIIa results in masking of high-affinity binding sites that are present in non-cross-linked fibrin. We propose that both plasmin and factor XIIIa play an important regulatory role in dissolution of blood clots by modulating t-PA-fibrin interaction.     

Glucocorticoid inhibition of urokinase-like plasminogen activators in cultured human lymphoblasts

Two human lymphoblast cell lines, LICR-LON-HMy2 (HMy2 cells) and GM4672A cells, are moderately growth inhibited by dexamethasone (1,4-pregnadien-9-fluoro-16 alpha-methyl-11 beta, 17 alpha, 21-triol-3,20-dione) (Dex). Both cell types secrete a urokinase (UK)-like plasminogen activator (PA). Treatment of both HMy2 and GM4672A cells with Dex for 1-4 days inhibits extracellular PA activity in a concentration-dependent manner, being half-maximal at approximately 1 X 10(-9)M. Inhibition of PA in both cell types is specific for active glucocorticoids, and this specificity parallels the ability of various steroids to bind to glucocorticoid receptors. HMy2 cell PA is fully suppressible by Dex, whereas up to one third of the activator expressed by GM4672A cells is resistant to glucocorticoid inhibition. Mixing experiments using a UK standard and conditioned media from Dex-treated cells suggest an absence of glucocorticoid-inducible inhibitors to UK or plasmin in both cell types. However, conditioned media from Dex-treated GM4672A cells inhibits a portion of the homologous cellular activator in conditioned media from control GM4672A cells. Thus, low levels of glucocorticoid-inducible inhibitors may contribute to, but cannot fully account for, Dex inhibition of GM4672A PA activity. Glucocorticoid-inducible inhibitors in HMy2 cells are either totally absent or are present at undetectable levels. Thus, regulation of UK-like PAs in HMy2 and GM4672A cells differs with respect to the extent to which glucocorticoids inhibit constitutively expressed activator levels, as well as the possible contribution of glucocorticoid-inducible inhibitors to the regulatory process in GM4672A cells.     

罗格列酮对高胰岛素培养的内皮细胞一氧化氮的影响及其机制研究

目的观察罗格列酮（RGZ）对高胰岛素培养的人脐静脉内皮细胞（HUVEC）NO浓度和内皮型一氧化氮合酶（eNOS）、磷酯酰肌醇3激酶（P13K）和蛋白激酶B（PKB）表达的影响,探讨RGZ改善高胰岛素状态下内皮功能障碍的信号转导机制。方法高浓度胰岛素培养HUVEC72h,并用不同浓度的RGZ进行干预。检测NO浓度,PI3K mRNA的表达,PKB、eNOS总蛋白和PKB丝氨酸473（PKB-Ser473）、eNOS丝氨酸1177（eNOS-Ser1177）的磷酸化表达。结果高浓度胰岛素培养HUVEC能呈剂帚和时间依赖性地降低N0的浓度,抑制内皮细胞P13KmRNA表达和PKB-Ser473、eNOS-Ser1177的磷酸化。用RGZ干预能硅著升高高胰岛素培养的内皮细胞NO的浓度和PKB、eNOS的磷酸化,增强PI3KmRNA表达;eNOS和P13K阻断剂均能阻断RGZ对高胰岛素培养的内皮细胞中NO浓度的升高,PI3K阻断剂还能阻断RGZ对高胰岛素培养内皮细胞PKB、eNOS的磷酸化。结论高胰岛素能下调P13K／PKB／eNOs信号通路而抑制内皮细胞NO的产生,RGZ能通过上调PI3K／PKB通路而增强高胰岛素培养的内皮细胞eNOS的活性和NO的产生。     

Thyroid follicular cells secrete plasminogen activators and can form angiostatin from plasminogen

Angiostatin, a 38 kDa fragment of plasminogen, potently inhibits the growth of blood vessels. Angiostatin is generated from plasminogen by urokinase-type (uPA) and tissue-type (tPA) plasminogen activators in the presence of free sulphydryl donors. Angiogenesis inhibitors may be important in regulating angiogenesis in developing goitre. We have examined angiostatin formation in human primary thyrocyte cultures and a rat thyrocyte cell line (FRTL-5). We found that human thyroid cells in culture secrete plasminogen activators (both tPA and uPA) as well as matrix metalloproteinase 2 into the medium. When human thyrocyte conditioned medium was incubated with plasminogen (10 microg/ml) and N-acetylcysteine (100 microM) for 24 h, a 38 kDa fragment of plasminogen, which is consistent with angiostatin, was generated. The appearance of the 38 kDa fragment was increased by agents that increase cAMP (forskolin and 8 BrcAMP). FRTL-5 cells, which do not secrete uPA or tPA, did not generate angiostatin. Thyroid cells produce several angiogenic growth factors, and human thyrocyte conditioned medium stimulated growth of endothelial cells. When the conditioned medium was incubated with plasminogen and N-acetylcysteine, this stimulatory effect was lost, consistent with the production of a growth inhibitory factor. We conclude that thyroid cells can produce angiostatin from plasminogen in vitro, and this may play a role in vivo in limiting goitre size.     

Interactions between live Bartonella bacilliformis and endothelial cells.

Bartonella bacilliformis, a gram-negative, flagellated, motile bacterium, is the etiologic agent of verruca peruana. It is found within the verruca, where it can form large cytoplasmic (Rocha-Lima) inclusions in endothelial cells. Previously, an activity has been described in homogenates of B. bacilliformis that in vitro increases the proliferation of endothelial cells and their production of tissue-type plasminogen activator (t-PA) and in vivo is angiogenic. The aim of the present study was to determine if live B. bacilliformis similarly stimulated endothelial cells and produced the Rocha-Lima inclusion. By measuring proliferation of cells and the production of t-PA in vitro, it was found that the live bacteria increased both parameters in a fashion similar to the homogenates of B. bacilliformis. Interaction between the bacteria and endothelial cells appeared to be necessary for proliferation. On electron microscopy, bacteria penetrated the endothelial cell within 1 h, forming a small membrane-bound inclusion. By 12 h, a large membrane-bound inclusion, similar to the Rocha-Lima inclusion, containing numerous bacteria was present. These data provide further evidence that B. bacilliformis has an angiogenic activity and that the bacteria are at least in part responsible for the vascular proliferation of the verruca.     

纤维蛋白原、纤维蛋白及其降解产物对共培养血管内皮细胞纤溶活性的影响

目的研究纤维蛋白原（Fg）、纤维蛋白（Fb）及其降解产物（FDP）对共培养体系中人脐静脉内皮细胞组织型纤溶酶原激活物和纤溶酶原激活物抑制剂表达的影响。方法应用Transwell膜建立人脐静脉内皮细胞-兔主动脉平滑肌细胞共培养体系，在不同浓度（0、0．5、1．5、3．0、4．5和6．0g／L）Fg、Fb和FDP干预24h后，分别检测该共培养体系中人脐静脉内皮细胞组织型纤溶酶原激活物和纤溶酶原激活物抑制剂mRNA水平（RT-PCR法）以及培养上清中组织型纤溶酶原激活物和纤溶酶原激活物抑制剂抗原含量（ELISA法）与活性（发色底物法）的变化情况。结果Fg对组织型纤溶酶原激活物的表达没有显著影响，较高浓度的Fg（3．0～4．5g／L）可明显促进纤溶酶原激活物抑制剂mRNA表达、抗原含量及活性升高，但过高浓度的Fg（6．0g／L）却抑制纤溶酶原激活物抑制剂的表达。3．0-4．5g／L的Fb对组织型纤溶酶原激活物mRNA和抗原含量都起上调作用，同时显著下调组织型纤溶酶原激活物活性。较高浓度的Fb（1．5-4．5g／L）则可明显上调纤溶酶原激活物抑制剂的表达，且在mRNA、蛋白和活性水平趋势基本一致。3．0-6．0g／L的FDP均可明显下调组织型纤溶酶原激活物mRNA、蛋白和活性水平，1．5-6．0mg／ml的FDP均可促进纤溶酶原激活物抑制剂的高表达。结论Fg、Fb和FDP可以通过影响组织型纤溶酶原激活物和纤溶酶原激活物抑制剂的表达，引起纤溶活性降低，参与动脉粥样硬化的发展进程。     

纤溶酶原激活物抑制剂-1反义RNA对主动脉内皮细胞部分功能的影响

目的 探讨纤溶酶原激活物抑制剂－1（plasminogen activator inhibior-1,PAI-1）反义RNA对离体培养的主动脉内皮细胞（endothelial cell,EC）PAI－1表达的作用及对血管内皮生长因子（vascular endothelial growth VEGF）的影响。方法 聚合酶链反应（PCR）手增PAI－1第2外显子,将PCR产物纯化克隆后连入真核细胞表达载体pcDNA3．1（－）,构建PAI－1反义RNA重组质粒。将pcDNA3．1－反义PAI－1重组质粒转染EC中。通过免疫组化、免疫印迹法（Westernblot）、双抗体夹心法（ELISA）检测细胞中PAI－1表达的改变,通过免疫荧光技术观察细胞中PAI－1表达量的变化对VEGF的影响。结果 转染后第3天,细胞中PAI－1含量为0．017ng/ml,细胞浆内代表VEGF的绿色荧光最弱,表明VEGF的表达也减少;第5天,PAI－1含量为0．093ng/ml,细胞浆内绿色荧光物质略增加。第7天,PAI－1含量为0．143ng/ml,接近正常值,VEGF的荧光染色接近于正常水平。结论 反义PAI－1RNA能有效阻断EC中PAI－1的蛋白合成,同时抑制剂细胞中VEGF的表达。     

Interactions between cultured bovine arterial smooth muscle cells and endothelial cells; studies on the release of growth inhibiting and growth stimulating factors

The balance of growth stimulating and growth inhibiting factors in the arterial wall might be of importance in the pathogenesis of atherosclerosis. A method using different dialysis steps was used to allow the simultaneous study of micromolecular (dialysable) and macromolecular (non-dialysable) substances in conditioned media from bovine and human arterial endothelial cells and smooth muscle cells in culture. Micromolecular substances inhibited the proliferation of aortic smooth muscle cells and endothelial cells, while the macromolecular substances were growth stimulating. The effect of the micromolecular and macromolecular factors was dose dependent, but only the micromoleculars were affected by conditioning time. The micromoleculars were heat stable. The effect of macromoleculars was completely abolished by heating to 100 degrees C for 5 minutes. Confluent cells released relatively more growth inhibiting and less growth stimulating activity while the balance was changed in subconfluent cells showing an increased release of growth stimulating activity per cell. A co-culture model for endothelial and smooth muscle cells demonstrated that the confluent aortic endothelial cells released relatively more growth inhibiting activity. These models seem suitable for the study of interactions of growth inhibition and stimulation between arterial cells in vitro in the normal or pathological state.     

The secretion of plasminogen activators by human myeloid leukemic cells in vitro

Peripheral blood cell preparation from 23 normal subjects and 72 patients with acute and 32 patients with chronic myeloid leukemia were cultured in vitro and released plasminogen activators were analyzed. The quantity of plasminogen activator secreted by leukemic cells varied widely and could not be correlated with the clinical severity of the disease. Immunochemical and electrophoretic techniques have been used to show that normal peripheral blood granulocytes released exclusively urokinase-like plasminogen activator, whereas leukemic cells secreted either urokinase or a tissue activator-like enzyme. The molecular species of enzyme released by acute myeloid leukemic cells may serve as a diagnostic marker of relevance to the management of this disease, since patients with acute myeloid leukemia whose cells released only tissue plasminogen activator did not respond to combination chemotherapy. Tissue plasminogen activators released by leukemic cells may display an unusual electrophoretic pattern that resembles that shown by urokinase. Immunochemical procedures are therefore essential for the correct identification of these enzymes.     

Injury produced by free fatty acids to lysosomes and mitochondria in cultured heart muscle and endothelial cells

Four-day primary coverglass cultures of rat heart muscle (M) and endothelioid (E) cells were treated for 30 min with stearic, oleic or linoleic acids in a FFA/albumin ratio of 6:1 at concentrations from 5 × 10⁻⁶ M to 5 × 10⁻⁴ M. Labilization of lysosomes and mitochondria was measured by acid phosphatase and succinic dehydrogenase staining activity respectively.

Stearate or linoleate, but not oleate, labilized M cell lysosomes at 5 × 10⁻⁶ M. Lysosomes of E cells were not significantly affected by any of the FFA at 5 × 10⁻⁴ M. The order of activity of these FFA for M cell lysosomes was LINOLEATE = stearate > oleate.

Both E and M cell mitochondria were significantly labilized by oleate or linoleate, 5 × 10⁻⁶ M, and by stearate, 5 × 10⁻⁵ M. The order of activity was linoleate > oleate > stearate.

Treatment of cultures for 24 hr with 50 μg/ml of hydrocortisone before the FFA at 5 × 10⁻⁵ M provided significant protection only against stearate-induced lysosomal labilization.