Acetyl-LDL stimulates macrophage-dependent plasminogen activation and degradation of extracellular matrix

The ability of acetyl-LDL to stimulate macrophage-dependent plasminogen activation and degradation of extracellular matrix was examined. We have found that expression of plasminogen activator activity in response to the scavenger receptor ligand varied among cell populations. Exposure to acetyl-LDL stimulated plasminogen activator expression by cells which constitutively released low levels of activator. These include a virally transformed macrophage-like cell line (RAW246.7), concanavalin A and C. parvum-activated macrophages. The stimulation of plasminogen activator activity was independent of cellular lipid accumulation since nonlipoprotein inhibitors of acetyl-LDL binding to the scavenger receptor stimulated activator expression in great excess to that observed with acetyl-LDL. In contrast, acetyl-LDL was unable to induce soluble plasminogen activator activity in cells which normally do not express it. These include a macrophage-like cell line (J774A.1) and resident peritoneal macrophages. Furthermore, acetyl-LDL was unable to modulate the copious secretion of activator by inflammatory macrophages elicited with thioglycolate. When macrophages were tested for their ability to degrade smooth muscle cell derived matrix, solubilization by resident, elicited, and activated cells was variously increased in the presence of plasminogen. Furthermore, exposure to acetyl-LDL enhanced plasmin-dependent degradation by resident cells and activated cells, whereas matrix degradation by elicited cells was unaffected.     

Developmental regulation of Sertoli cell aromatase activity and plasminogen activator production by hormones, retinoids and the testicular paracrine factor, PModS.

Testicular peritubular cells produce a paracrine factor termed PModS that has dramatic effects on Sertoli cell function in vitro. The current study was designed to examine the actions of PModS and hormones on Sertoli cell aromatase activity and plasminogen activator production at various stages of pubertal development. Sertoli cells were isolated from 10-, 20-, and 35-day-old rats (ages correspond to prepubertal, midpubertal, and late-pubertal stages of development). Aromatase activity was found to be high and hormone-responsive in prepubertal Sertoli cells and to decline and be nonresponsive to hormones in late-pubertal Sertoli cells. FSH was the only hormone found to influence aromatase activity and estrogen production. PModS alone was not found to affect aromatase activity at any of the developmental stages examined. Interestingly, PModS was found to suppress the ability of FSH to stimulate aromatase activity and estrogen production in midpubertal Sertoli cells. Results imply that PModS may promote Sertoli cell differentiation to a more adult stage of development that is less responsive to FSH in stimulating aromatase activity. In contrast to aromatase activity, plasminogen activator production was found to increase during pubertal development. Production of Sertoli cell tissue-type plasminogen activator (tPa) was stimulated by FSH at each of the developmental stages examined, whereas production of urokinase-type plasminogen activator (uPa) was influenced by FSH only in prepubertal Sertoli cells. Insulin also stimulated uPa and tPa production by prepubertal Sertoli cells, and retinol significantly suppressed uPa production and the ability of FSH to stimulate tPa production by midpubertal Sertoli cells.     

Heparin stimulation of plasminogen activator secretion by macrophage-like cell line RAW264.7: role of the scavenger receptor

Secretion of urokinase-type plasminogen activator (uPA) by RAW264.7 cells was stimulated by heparin in a dose- and time-dependent manner. Secretion of uPA was not detected when cells were exposed to heparin at 4 degrees C, indicating that heparin was not simply releasing receptor-bound uPA. Furthermore, prior removal of membrane-associated uPA did not influence heparin's ability to stimulate the release of uPA from the macrophage-like line. Low molecular weight and weakly anticoagulant heparins stimulated uPA secretion but less effectively than other heparin fractions. The observed stimulation in macrophage uPA secretion by heparin is similar to that previously reported for polyanions recognized by the scavenger receptor including fucoidan, polyinosinic acid, dextran sulfate, and acetyl-LDL (Falcone and Ferenc: J. Cell. Physiol., 135:387-396, 1988). Evidence that heparin's binding to RAW264.7 cells is mediated by the scavenger receptor is derived from experiments in which fucoidan blocked the specific binding of [3H]-heparin to RAW264.7 cells. However, heparin partially inhibited the stimulation of cholesteryl [3H]-oleate synthesis observed in these cells upon incubation with acetyl-LDL and weakly inhibited cellular binding of 125I-acetyl-LDL at 4 degrees C. These data indicate that heparin's binding to RAW264.7 cells is mediated, only in part, by the scavenger receptor. Nonetheless, neither heparin nor fucoidan was able to stimulate the release of plasminogen activator activity from monocyte-like U937 cells which are devoid of scavenger receptor activity.     

Effect of interleukin-10 on the Paracoccidioides brasiliensis killing by gamma-interferon activated human neutrophils

Paracoccidioidomycosis, a deep mycosis endemic in Latin America, is a chronic granulomatous disease caused by the fungus Paracoccidioides brasiliensis. Phagocytic cells play a critical role against this fungus, and several studies have shown the effects of activator and suppressive cytokines on macrophage and monocyte functions. However, studies on polymorphonuclear neutrophils (PMNs), that are the first cells recruited to the infection sites, are scarcer. Thus, the objective of this paper was to assess whether interleukin-10 (IL-10), a potent anti-inflammatory cytokine, is able to block the activity of IFN-gamma-activated human PMNs upon P. brasiliensis intracellular killing, in vitro. The results showed that IFN-gamma-activated PMNs have an effective fungicidal activity against the fungus. This activity was associated with the release of high levels of H(2)O(2), the metabolite involved in phagocytic cells antifungal activities. However, the concomitant incubation of these cells with IFN-gamma and IL-10 significantly blocked IFN-gamma activation. As a consequence, PMNs killing activity and H(2)O(2) release were inhibited. Together, our results show the importance of PMNs exposure to activator or suppressor cytokines in the early stages of paracoccidioidomycosis infection.     

Arsenic trioxide (As2O3) inhibits invasion of HT1080 human fibrosarcoma cells: role of nuclear factor-kappaB and reactive oxygen species

In order to define the role of As2O3 in regulating the tumor cell invasiveness, the effects of As2O3 on secretion of matrix metalloproteinases (MMPs) and urokinase plasminogen activator (uPA), and in vitro invasion of HT1080 human fibrosarcoma cells were examined. As2O3 inhibited cell adhesion to the collagen matrix in a concentration dependent manner, whereas the same treatment enhanced cell to cell interaction. In addition, As2O3 inhibited migration and invasion of HT1080 cells stimulated with phorbol 12-myristate 13-aceate (PMA), and suppressed the expression of MMP-2, -9, membrane type-1 MMP, uPA, and uPA receptor (uPAR). In contrast, As2O3 increased the expression of tissue inhibitor of metalloproteinase (TIMP)-1 and PA inhibitor (PAI)-1, and reduced the MMP-2, -9, and uPA promoter activity in the presence and absence of PMA. Furthermore, the promoter stimulating and DNA binding activity of nuclear factor-kappaB (NF-kappaB) was blocked by As2O3, whereas the activator protein-1 activity was unchanged. Pretreatment of the cells with N-acetyl-L-cysteine (NAC) significantly prevented suppression of MMPs and uPA secretion, DNA binding activity of NF-kappaB, and in vitro invasion of HT1080 cells by As2O3, suggesting a role of reactive oxygen species (ROS) in this process. These results suggest that As2O3 inhibits tumor cell invasion by modulating the MMPs/TIMPs and uPA/uPAR/PAI systems of extracellular matrix (ECM) degradation. In addition, the generation of ROS and subsequent suppression of NF-kappaB activity by As2O3 might partly be responsible for the phenomena. Overall, As2O3 shows potent activity controlling tumor cell invasiveness in vitro.     

三七皂苷Rg1对tPA和PAI-1活性的调节作用

探讨三七皂苷Rg1对组织型纤溶酶原激活物(tPA)和纤溶酶原激活物抑制物(PAI-1)活性的调节作用。运用发色底物方法测定三七皂苷Rg1在体外和静脉注射对家兔血浆纤溶酶原激活物(tPA)和血浆或血小板释放的纤溶酶原激活物抑制物(PAI-1)水平的影响。结果表明,三七皂苷Rg1在体外呈浓度依赖性明显抑制血浆PAI-1活性,同时提高血浆tPA活性;30和60 mg/kg的三七皂苷Rg1静脉注射显著抑制血浆PAI-1活性,提高血浆tPA活性,同时降低凝血酶激活的血小板所释放的PAI-1水平。本实验提示三七皂苷Rg1能抑制PAI-1活性,同时升高tPA活性可能是其抗血栓作用的分子机制之一。     

Oxygen modulates the release of urokinase and plasminogen activator inhibitor-1 by retinal pigment epithelial cells

The aims of this study were to examine the effect of oxygen, in the presence or absence of exogenous growth factors, on the release of plasminogen activators and plasminogen activator inhibitor-1 by cultured human retinal pigment epithelial cells. Antigen and activity levels of urokinase, tissue plasminogen activator and plasminogen activator inhibitor were measured in conditioned media after cells were exposed to three different oxygen environments: hypoxia, normoxia and hyperoxia. Overall proteolytic balance was determined by zymography. The effects of exogenous basic fibroblast growth factor and transforming growth factor-beta were also examined. it was found that retinal pigment epithelial cells released urokinase, tissue plasminogen activator and plasminogen activator inhibitor in measurable quantities. After 48 h, urokinase levels were highest at normoxia, reaching 7.2ng/10(6) cells (+/-2.0 SEM), whereas plasminogen activator inhibitor 1 levels were highest at hyperoxia, reaching 67.5ng/10(6) cells (+/-3.7 SEM). Tissue plasminogen activator levels were minimal (<0.5ng/10(6) cells) and unaffected by both oxygen and growth factors. Overall proteolytic activity was also greatest at normoxia. Fibroblast growth factor stimulated urokinase production dose-dependently, but plasminogen activator inhibitor only minimally. Transforming growth factor-beta stimulated plasminogen activator inhibitor production dose-dependently but urokinase only at higher concentrations. These results suggest that both oxygen tension and growth factors may interact to modulate the proteolytic properties of the human retinal pigment epithelium.     

Oligodeoxyribonucleotides derived from salmon sperm DNA: An alternative to defibrotide

Defibrotide is a single-stranded nucleic acid polymer originally derived from porcine mucosa. Cheap salmon sperm DNA is commercially available and widely used in drug production. In this study, oligodeoxyribonucleotides were successfully obtained from the controlled depolymerization of salmon sperm DNA. The obtained product shared similar chemical and biological properties with defibrotide produced by Gentium SpA, Italy. It was also found that oligodeoxyribonucleotides derived from non-mammalian origins could also directly stimulate tissue plasminogen activator (t-PA) release from cultured human endothelial cells, and enhance fibrinolytic activity in the rabbit.     

Inhibition of endothelial cell proliferation by the recombinant kringle domain of tissue-type plasminogen activator

Tissue-type plasminogen activator (tPA) is a multidomain serine protease that converts the zymogen plasminogen to plasmin. tPA contains two kringle domains which display considerable sequence identity with those of angiostatin, an angiogenesis inhibitor. TK1-2, a recombinant kringle domain composed of t-PA kringles 1 and 2 (Ala(90)-Thr(263)), was produced by both bacterial and yeast expression systems. In vitro, TK1-2 inhibited endothelial cell proliferation stimulated by basic fibroblast growth factor, vascular endothelial growth factor, and epidermal growth factor. It did not inhibit proliferation of non-endothelial cells. TK1-2 also inhibited in vivo angiogenesis in the chick embryo chorioallantoic membrane model. These results suggest that the recombinant kringle domain of t-PA is a selective inhibitor of endothelial cell growth and identifies this molecule as a novel anti-angiogenic agent.     

Human monocyte or recombinant interleukin 1's are specific for the secretion of a metalloproteinase from chondrocytes

Macrophage products induce production of proteases that contribute to cartilage degradation in various joint diseases. In these studies we stimulated rabbit chondrocytes with various cytokines in vitro in order to determine which were responsible for changes in the release of prostaglandin, plasminogen activator, and a metalloproteinase. The metalloproteinase assayed in these studies is a latent enzyme whose activity can rapidly be measured with fluorogenic casein. Conditioned media from stimulated human peripheral blood mononuclear cells; purified human monocyte IL 1, pI 7,6, and 5; and recombinant human IL 1, beta or alpha forms, all changed the secretory pattern of rabbit articular chondrocytes in a similar manner: production and secretion of a latent metalloproteinase(s) and prostaglandin E were stimulated in a concentration-dependent fashion, whereas the activity of plasminogen activator was strongly reduced. Antibodies against human monocyte IL 1 blocked the active principle in various mononuclear cell-conditioned media, suggesting that uncharacterized factors present in these supernatants do not affect the metalloproteinase response. When added to confluent chondrocytes, phorbol myristate acetate, concanavalin A, IL 2, lipopolysaccharide, indomethacin, and prostaglandin E2, which interfere with lymphocyte proliferation assays for IL 1, failed to influence chondrocyte metalloproteinase secretion. Recombinant human IFN-alpha or IFN-gamma in the presence or absence of IL 1 had no effect on rabbit chondrocytes, whereas recombinant human tumor necrosis factor decreased plasminogen activator but had no effect on prostaglandin or metalloproteinase production. These results support the concept that IL 1 specifically induces chondrocytes to produce metalloproteinases, and hence may play an important role in destructive joint diseases.     

Increased proteolytic activity is responsible for the aberrant morphogenetic behavior of endothelial cells expressing the middle T oncogene

Expression of the polyoma virus middle T (mT) oncogene in vivo is associated with a profound subversion of normal vascular development, which results in the formation of endothelial tumors (hemangiomas). In an attempt to understand the molecular mechanisms responsible for this phenomenon, we have investigated, in an in vitro system, the morphogenetic properties of endothelial cells expressing this oncogene. mT-expressing endothelioma (End) cells grown within fibrin gels formed large hemangioma-like cystic structures. All End cell lines examined expressed high levels of fibrinolytic activity resulting from increased production of urokinase-type plasminogen activator and decreased production of plasminogen activator inhibitors. Neutralization of excess proteolytic activity by exogenously added serine protease inhibitors corrected the aberrant in vitro behavior of End cells and allowed the formation of capillary-like tubules. These results suggest that tightly controlled proteolytic activity is essential for vascular morphogenesis and that physiological protease inhibitors play an important regulatory role in angiogenesis.     

Concomitant secretion by transformed SVWI38-VA13-2RA cells of plasminogen activator(s) and substance (s) which prevent their detection.

SVWI38-VA13-2RA cells have been shown to secrete both plasminogen activator(s) and inhibitory substance(s) which prevent detection of plasminogen activator activity in the widely used ¹²⁵I-labeled fibrin dish assay. The SVWI38-VA13-2RA plasminogen activator(s) can be detected by assay of individual gel slices following SDS gel electrophoresis of SVWI38-VA13-2RA cell conditioned medium. The inhibitory substance(s) have been detected by the ability of SVWI38-VA13-2RA conditioned medium to inhibit the activity of mouse lung carcinoma (CMT64) cell plasminogen activator(s). Thus, lack of plasminogen activator activity with the ¹²⁵I-labeled fibrin dish assay alone no longer suffices as proof that cells are not secreting plasminogen activator(s). Concomitant secretion of plasminogen activators and inhibitors must be assessed in attempts to correlate viral transformation, tumorigenicity and plasminogen activator levels.     

Antithrombotic and profibrinolytic activities of eckol and dieckol

In order to develop new anticoagulant agents, two single compounds (eckol and dieckol) were isolated from Eisenia bicyclis and examined their anticoagulant activities by monitoring activated partial thromboplastin time (aPTT), prothrombin time (PT) as well as cell-based thrombin and activated factor X (FXa) generation activities. And the effects of eckol and dieckol on the expression of plasminogen activator inhibitor type 1 (PAI-1) and tissue-type plasminogen activator (t-PA) were tested in tumor necrosis factor-α (TNF-α) activated human umbilical vein endothelial cells (HUVECs). Data showed that eckol and dieckol prolonged aPTT and PT significantly and inhibited thrombin and FXa activities. They also inhibited the generation of thrombin or FXa in HUVECs. In accordance with these anticoagulant activities, eckol or dieckol showed anticoagulant effect in vivo. Furthermore, eckol and dieckol inhibited TNF-α induced PAI-1 production and the ratio between PAI-1 and t-PA was found to be significantly decreased by eckol and dieckol. Surprisingly, these anticoagulant and profibrinolytic effects of dieckol were better than those of eckol indicating that hydroxyl group in eckol positively regulated anticoagulant function of eckol. Therefore, these results suggest that eckol or dieckol possesses antithrombotic activities and provides a possibility to develop as an agent for the anticoagulation.     

Hepatocyte growth factor modulates migration and proliferation of human microvascular endothelial cells in culture.

Epidermal growth factor (EGF) induces tubular formation of cultured human omental microvascular endothelial (HOME) cells and EGF also stimulates cell migration as well as expression of tissue type plasminogen activator (t-PA). Here we studied the effects of hepatocyte growth factor (HGF) on cell proliferation, cell migration and expression of t-PA and other related genes. Migration of confluent HOME cells into the denuded space was stimulated by HGF after being wounded with razor blade, but at a reduced rate in comparison with EGF. HOME cells could be proliferated in response to exogenous 100 ng/ml of HGF at rates comparable to that of 20 ng/ml EGF. The chemotactic activity of HOME cells was significantly stimulated by HGF in a dose-dependent manner when assayed by Boyden chamber. HGF did not efficiently enhance expression of both the t-PA gene and a tissue inhibitor of metalloproteinase gene whereas it stimulated expression of plasminogen activator inhibitor-1. Our present study provides a new evidence that some of the biological effects of HGF on HOME cells in culture are similar to those of EGF.     

The effects of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) on human osteoblast-like cells

The activity of human osteoblast-like cells cultured in vitro is regulated by a number of factors, which include systemic hormones as well as agents that can be produced locally within bone. Several cytokines and growth factors have been demonstrated to be produced by osteoblasts themselves, and this includes granulocyte-macrophage colony-stimulating factor (GM-CSF). In this report we show that recombinant human GM-CSF (rhGM-CSF) modulates the activities of osteoblast-like cells derived from human trabecular bone in vitro. rhGM-CSF stimulated the proliferation of the cultured human osteoblast-like cells, but antagonised the induction by 1,25(OH)2D3 of osteocalcin synthesis and alkaline phosphatase activity, two characteristic products of osteoblasts. rhGM-CSF however, had no appreciable effect on the production of prostaglandin E2, or on the plasminogen activator activity associated with human osteoblast-like cells. These results are the first report of which we are aware of an apparently direct action of GM-CSF on cells of the osteoblast phenotype. These studies indicate that GM-CSF represents another haematological factor that can potentially exert regulatory actions on human osteoblast-like cells. GM-CSF may therefore be a potential paracrine/autocrine regulator of osteoblast activity.     

Purification of c-phycocyanin from Spirulina fusiformis and its effect on the induction of urokinase-type plasminogen activator from calf pulmonary endothelial cells

c-Phycocyanin (c-pc), a blue coloured, fluorescent protein was purified from blue-green alga, Spirulina fusiformis and its effect on fibrinolytic system in vascular endothelial cells was investigated.The c-pc consisted of two subunits, alpha and beta, whose molecular masses were 16 and 17 kDa, respectively. N-terminal sequences of both subunits were well conserved compared with other blue green algal phycobiliproteins. Fibrinolytic activity in the medium conditioned by calf pulmonary arterial endothelial cells was measured by the fibrin plate method.The c-pc increased the fibrinolytic activity in dose- and time-dependent manners. Fibrin zymographic studies indicated that c-pc-induced urokinase-type plasminogen activator in the cells. These in vitro results suggest that c-pc from S. fusiformis is a potent profibrinolytic protein in the vascular endothelial system.     

Stimulation of plasminogen activator production by dimethyl sulfoxide in Chinese hamster ovary cells

The production of plasminogen activator activity in an auxotrophic mutant of the Chinese hamster ovary cell line was found be greatly stimulated by low concentrations of dimethyl sulfoxide. The production of both cell-associated and excreted plasminogen activator activities was stimulated maximally by dimethyl sulfoxide at a concentration of 2.5%. The stimulation of plasminogen activator activity production was found to be completely inhibited by actinomycin D and cycloheximide but not by mitomycin C, implying that new protein and RNA syntheses were required for this process. Using specific antibodies against plasminogen activator, the presence of a tissue-type plasminogen activator could only be detected in dimethyl sulfoxide treated cells. The dimethyl sulfoxide induced plasminogen activator production was observed only in a mutant auxotrophic for adenosine, glycine, and thymidine but not in wild-type cells. The ability of dimethyl sulfoxide to induce the synthesis of plasminogen activator was lost when the cells were hybridized with another complementary auxotrophic mutant. This implies that the ability of dimethyl sulfoxide to stimulate the production of plasminogen activator may be related to the auxotrophic mutation in this cell.     

Heparin potentiation of 3T3-adipocyte stimulated angiogenesis: mechanisms of action on endothelial cells

We have examined the cellular mechanisms by which heparin potentiates the ability of 3T3-adipocytes to stimulate the formation of new blood vessels. Both anticoagulant and non-anticoagulant heparin species enhanced the angiogenic activity of adipocyte-secreted products in the chick chorioallantoic membrane assay, indicating that the angiotropic effect of this glycosaminoglycan is independent of its effect on the coagulation cascade. Heparin alone was unable to produce a neovascular response. The ability of heparin to modulate three endothelial functions in vitro thought to be related to angiogenesis were examined: protease activity, motility, and mitogenesis. Heparin caused a 100% increase in the adipocyte-induced stimulation of endothelial cell plasminogen activator activity and motility, but had no effect on proliferation. The enhancement of plasminogen activator and chemoattractant activities had a similar ED50 (1-2 micrograms/ml) and optimum dose (10-30 micrograms/ml). When we examined the direct effect of heparin on the activity of two distinct plasminogen activator enzymes--urokinase and tissue-type--a dual action of heparin was observed: tissue-type enzyme activity was stimulated 100% by heparin at 10 micrograms/ml, whereas urokinase activity was inhibited by 77% at this dose. These data suggest that heparin potentiates angiogenesis in vivo by stimulating endothelial cell plasminogen activator, motility, or both. Our results further suggest that for adipocyte-induced blood vessel formation, in contrast to other angiogenesis systems, heparin does not appear to affect the mitogenic activity.     

Transduced endothelial cells expressing high levels of tissue plasminogen activator have an unaltered phenotype in vitro

Elevated cellular plasminogen activator activity has been associated with significant alterations in the in vitro phenotype of both malignant cell lines and nonmalignant endothelial cells. To examine the role of elevated cellular plasminogen activator activity in the production of altered endothelial cell behavior, bovine coronary artery endothelial cells were transduced with a retroviral vector expressing large amounts of tissue plasminogen activator. Cells transduced with the tissue plasminogen activator vector were compared with both untransduced cells and cells transduced with a control vector in a series of in vitro assays of cellular attachment, proliferation, migration, and invasion. The morphology of the 2 transduced populations was unchanged. There was a small decrease (5–15%) in the horizontal migration rate of both transduced cell populations, as compared with that of untransduced cells. No significant differences were detected among the three cell populations in any of the other assays. We conclude that expression of high levels of tissue plasminogen activator does not specifically affect endothelial cell phenotype in vitro. These data lend support to the hypothesis that elevated plasminogen activator activity is necessary but not sufficient to produce alterations in endothelial cell behavior. © 1993 Wiley-Liss, Inc.     

Statin Drugs and Dietary Isoprenoids Downregulate Protein Prenylation in Signal Transduction and Are Antithrombotic and Prothrombolytic Agents

Statins and various isoprenoids of dietary origins inhibit L-mevalonic acid synthesis, which in turn downregulates cholesterol and various other dependent substances, including farnesyl- and geranylgeranyl-conjugated proteins involved in cell signaling processes. Such signaling processes are stimulated by protease activated receptor-1 (PAR-1), which upon activation, causes the expression of various substances including tissue factor (TF) and plasminogen activator inhibitor-1 (PAI-1). Tissue factor promotes thrombin generation, where thrombin stimulates a variety of cellular processes, as well as activating PAR-1 to produce more thrombin. Statins downregulate TF mitigating thrombin generation and also downregulate PAI-1, which normally consumes tissue plasminogen activator (tPA). In the absence of PAI-1, tPA activates plasminogen to generate plasmin. Thus, statins behave as antithrombotic agents and prothrombolytic agents.