Plasmin-mediated macrophage reversal of low density lipoprotein aggregation

Evidence suggests that aggregated low density lipoprotein (AgLDL) accumulates in atherosclerotic lesions. Previously, we showed that AgLDL induces and enters surface-connected compartments (SCC) in human monocyte-derived macrophages by a process we have named patocytosis. Most AgLDL taken up by these macrophages in the absence of serum is stored in SCC and remains undegraded. We now show that macrophages released AgLDL (prepared by vortexing or treatment with phospholipase C or sphingomyelinase) from their SCC when exposed to 10% human lipoprotein-deficient serum (LPDS). Macrophages also took up AgLDL in the presence of LPDS, but subsequently released it. In both cases, the released AgLDL was disaggregated. Although the AgLDL that macrophages took up could not pass through a 0.45-micrometer filter, >60% of AgLDL could pass this filter after release from the macrophages. Disaggregation of AgLDL was verified by gel-filtration chromatography and electron microscopy that also showed particles larger than LDL, reflecting fusion of LDL that aggregates. The factor in serum that mediated AgLDL release and disaggregation was plasmin generated from plasminogen by macrophage urokinase plasminogen activator. AgLDL release was decreased >90% by inhibitors of plasmin (epsilon-amino caproic acid and anti-plasminogen mAb), and also by inhibitors of urokinase plasminogen activator (plasminogen activator inhibitor-1 and anti-urokinase plasminogen activator mAb). Moreover, plasminogen could substitute for LPDS and produce similar macrophage release and disaggregation of AgLDL. Because only plasmin bound to the macrophage surface is protected from serum plasmin inhibitors, interaction of AgLDL with macrophages was necessary for reversal of its aggregation by LPDS. The released disaggregated LDL particles were competent to stimulate LDL receptor-mediated endocytosis in cultured fibroblasts. Macrophage-mediated disaggregation of aggregated and fused LDL is a mechanism for transforming LDL into lipoprotein structures size-consistent with lipid particles found in atherosclerotic lesions.     

Close similarity between cultured human omental mesothelial cells and endothelial cells in cytochemical markers and plasminogen activator production

Summary The mesothelial cells obtained from human omental adipose tissue showed a typical cobblestone monolayer and reacted strongly with keratin, but did not have Von Willebrand factor. Ultrastructurally these cells revealed the existence of desmosome-like cell junctions as well as intracellular canaliculi, tubular structures surrounded by microvilli, and tonofilament-like filaments. The mesothelial cells grew much faster in the medium containing epidermal growth factor, actively took up acetylated-low density lipoprotein into their cytoplasm, and released angiotensin-converting enzyme. They also released urokinase-type plasminogen activator, but only half as much as do human umbilical vein endothelial cells; release of tissue-type plasminogen activator was not observed. Inasmuch as the mesothelial cells also released plasminogen activator inhibitor-1, as do human umbilical vein endothelial cells, we could not detect u-PA activity in culture medium. u-PA may play a role in the protection against adhesion among visceral organs. These observations indicate that cultured human mesothelial cells have characteristics closely related to those found in human endothelial cells.     

Human urokinase contains GalNAc beta (1-4)[Fuc alpha (1-3)]GlcNAc beta (1-2) as a novel terminal element in N-linked carbohydrate chains.

Structural analysis of enzymically released N-linked carbohydrate chains of human urokinase (urinary-type plasminogen activator) by 1H NMR spectroscopy and FAB-MS demonstrated that the N-linked oligosaccharides on the only N-glycosylation site contain diantennary structures with the novel GalNAc beta (1-4) [Fuc alpha (1-3)]GlcNAc beta (1-2) element in the upper or the lower branch.     

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.     

嵌合分子ut-PA(1)sf-9表达产物的纯化及其性质的初步鉴定

嵌合蛋白 ut- PA( u- PA与 t- PA的嵌合型纤溶酶原激活剂 )基因删除编码 u- PA中 R1 78—R1 81的 1 2个核苷酸后 ,得到的嵌合分子 ut- PA( 1 )在昆虫细胞 sf- 9中表达 ,通过苯甲脒 -Sepharose 6B亲和柱层析对表达产物进行纯化 .得到的纯品走 SDS- PAGE,结果显示 ,其分子量为60 k D.血凝块溶解实验结果表明 ,嵌合分子 ut- PA( 1 )有良好的体外溶栓能力 .在不同剂量 PAI- 1的抑制条件下测定 ,亲本嵌合蛋白 ut- PA的活力分别下降 2 2 .7%和 1 3.8%时 ,嵌合分子 ut- PA( 1 )只分别下降了 1 2 .9%和 9.1 % .说明突变体 ut- PA( 1 )具有一定的对 PAI- 1抑制作用的抗性     

The stimulation of human synovial fibroblast plasminogen activator activity. Involvement of cyclic AMP and cyclooxygenase products

The plasminogen activator activity of human synovial fibroblasts is raised by a monocyte-derived polypeptide, synovial activator and also by all-trans retinoic acid. The elevation of the synovial cell plasminogen activator activity by the two stimuli is potentiated both by agents which can raise cellular cyclic AMP levels, namely prostaglandin E2, cholera toxin and 3-isobutyl-1-methylxanthine, and also by exogenous 8-bromocyclic AMP. These findings suggest that there might be a substrate, which is phosphorylated by a cyclic AMP-dependent protein kinase and which is important in the modulation of the synovial cell plasminogen activator activity by the two stimuli. Prostanoids can be important in the stimulation of the synovial fibroblast plasminogen activator activity by mononuclear cell supernatants, since indomethacin can inhibit the increase in proteinase activity.     

Isolation of plasminogen activator from human plasma by chromatography on lysine-sepharose.

A plasminogen activator has been recovered from human postexercise blood plasma by chromatography on lysine coupled to Sepharose 4B. Plasminogen activator activity was unstable in citrated plasma but could be stabilized by addition of 5 mm EDTA and 5 mm benzamidine. Stabilized postexercise plasma was applied to lysine-Sepharose and the bulk of the protein washed from the column with a gradient in NaCl (0–0.6 m). The activator was then eluted with 1.5 m NaCl or with linear gradients in l-lysine (0–20 mm) or l-arginine (0–0.2 m). The activator was purified approximately 5000-fold over the starting plasma and was well separated from plasminogen and plasmin inhibitors. It caused no lysis on plasminogenfree fibrin-agar plates. The activator activity was inhibited by diisopropylphosphorofluoridate, ?-aminocaproic acid, and lysine but not by soybean trypsin inhibitor, Trasylol or blood plasma with or without heparin present. A plasminogen activator isolated from homogenized human vein by identical chromatography on lysine-Sepharose showed the same inhibition profile. This suggested that the activator recovered from postexercise human plasma was released from the blood vessel walls. Incorporation studies with [³²P]-diisopropylphosphorofluoridate indicated that the enzyme displays over 30,000 Committee on Thrombolytic Agents units/mg of protein. This high potency combined with the activator's resistance to plasma inhibitors suggests that it may be of importance to in vivo fibrinolysis.     

Influence of macrophage products on the release of plasminogen activator, collagenase, beta-glucuronidase and prostaglandin E2 by articular chondrocytes.

We describe the effects of products of mononuclear phagocytes on the secretory activity of chondrocytes. The primary confluent cultures of rabbit articular chondrocytes were exposed to standard medium alone or enriched with conditioned medium obtained from cultures of rabbit peritoneal macrophages, the mouse macrophage cell line P388D1 or human blood mononuclear cells. Four markers of release were assessed, the neutral proteinases plasminogen activator and collagenase, the acid hydrolase beta-glucuronidase and prostaglandin E2, and the kinetics of their changes were monitored. Chondrocytes that were cultured in standard medium secreted large amounts of plasminogen activator, some beta-glucuronidase, but no collagenase, and released only minor amounts of prostaglandin E2. The addition of conditioned medium from rabbit macrophages induced a rapid release of large quantities of prostaglandin E2 and an abundant secretion of collagenase, while abolishing or strongly decreasing plasminogen activator secretion. In addition, beta-glucuronidase secretion was markedly enhanced. The decrease in secretion of plasminogen activator appeared to reflect a diminished production, since no evidence was found for the generation of inhibitors or for an accelerated extracellular breakdown of the enzyme. Conditioned media of the mouse and human mononuclear cells influenced the secretory activities of rabbit articular chondrocytes in a similar way, suggesting that the factor (or factors) acting on chondrocytes is produced by a variety of macrophages, and that its action is not species-restricted. The time course and concentration-dependence of the effects observed indicate that the secretion of plasminogen activator and collagenase are influenced in a strictly reciprocal fashion by the macrophage products. The release of prostaglandin E2 paralleled that of collagenase.     

Fluorometric high-performance liquid chromatography of N-acetyl- and N-glycolylneuraminic acids and its application to their microdetermination in human and animal sera, glycoproteins, and glycolipids

A simple, rapid, and highly sensitive high-performance liquid chromatographic method is described for the determination of N-acetyl- and N-glycolylneuraminic acids in human and animal sera, glycoproteins, and glycolipids. The neuraminic acids, released by acid hydrolysis of these biological samples, are converted in dilute sulfuric acid with 1,2-diamino-4,5-methylene-dioxybenzene, a fluorogenic reagent for alpha-keto acids, to highly fluorescent derivatives. The derivatives are separated within 12 min on a reversed-phase column (Radial-Pak cartridge C18) with an isocratic elution and detected fluorometrically. The detection limits are 25 fmol (7.7 pg) for N-acetylneuraminic acid and 23 fmol (7.5 pg) for N-glycolylneuraminic acid in a 10-microliter injection volume at a signal-to-noise ratio of 2. This method permits precise determination of the neuraminic acids in 5 microliter of human and animal sera or in 0.25-2.5 micrograms of glycoproteins and glycolipids.     

Importance, localization and functional properties of the cell-associated form of plasminogen activator in mouse peritoneal macrophages

In inflammatory macrophages, plasminogen activator exists in two active forms, a soluble form released into the extracellular medium and a cell-associated form. This communication describes some properties of the cellular form of plasminogen activator, in intact macrophages and in cell lysates. Cellular plasminogen activator is a membrane protein, associated with the outer face of the plasma membrane; in intact macrophages, it participates in the activation of exogenous plasminogen and, thus, has to be considered as an ectoenzyme. A plasminogen activator activity can be detected in cell lysates (macrophage monolayers lysed in 0.1% Triton X-100) only when plasmin production is followed by the use of small synthetic substrates because a soluble inhibitor, released during extraction, blocks plasmin fibrinolytic activity. In these lysates, plasminogen activator molecules exist as high molecular weight unstable complexes exhibiting a high affinity for plasminogen.     

Purification and characterization of single-chain urokinase-type plasminogen activator from human cell cultures

A urokinase-type plasminogen activator was purified from conditioned media of several human cell cultures, but preferably from the human lung adenocarcinoma line CALU-3 (ATCC, HTB-55), using a combination of chromatography on zinc chelate-Sepharose, SP-Sephadex C-50, and Sephadex G-100. Final yields of 65-100 micrograms/liter of starting material were obtained with a 290-fold purification factor and a recovery of 30%. The purified plasminogen activator consists of a single polypeptide chain with Mr 54,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and is very similar or identical to single-chain urokinase-type plasminogen activator on the basis of immunodiffusion, amino acid composition, and the lack of specific binding to fibrin. It has very low amidolytic activity on Pyroglu-Gly-Arg-rho-nitroanilide and is converted to two-chain urokinase by limited exposure to plasmin. It has a specific activity of 60,000 IU/mg on fibrin plates and directly activates plasminogen following Michaelis-Menten kinetics with Km = 1.1 microM and kappa cat = 0.0026 S-1. It is concluded that the plasminogen activator purified from CALU-3-conditioned media is physically and kinetically identical to single-chain urokinase-type plasminogen activator. With the present straightforward purification method and a readily available source, sufficient amounts of single-chain urokinase-type plasminogen activator can be obtained for more detailed investigations of its biochemical, biological, and thrombolytic properties.     

Establishment of a humanized model of liver using NOD/Shi-scid IL2Rgnull mice

Severely immunodeficient NOD/Shi-scid IL2Rg^null (NOG) mice are used as recipients for human tissue transplantation, which produces chimeric mice with various types of human tissue. NOG mice expressing transgenic urokinase-type plasminogen activator in the liver (uPA-NOG) were produced. Human hepatocytes injected into uPA-NOG mice repopulated the recipient livers with human cells. The uPA-NOG model has several advantages over previously produced chimeric mouse models of human liver: (1) the severely immunodeficient NOG background enables higher xenogeneic cell engraftment; (2) the absence of neonatal lethality enables mating of homozygotes, which increased the efficacy of homozygote production; and (3) donor xenogeneic human hepatocytes could be readily transplanted into young uPA-NOG mice, which provide easier surgical manipulation and improved recipient survival.     

Release of plasminogen activator by pentosan polysulfate

In isolated perfused organs (pig ear, rabbit ear, rat lung) pentosan polysulphate caused an increase in the release of plasminogen activator. The activator was released in a dose-dependent manner, the release being repeatedly induced as demonstrated with the rabbit ear. An increase in activator activity was also found in experimental animals (mini-pig, rat, rabbit). In the isolated perfused organ and the whole animal, the activator released proved to be tissue-type plasminogen activator. For the release mechanism displacement of mural plasminogen activator by pentosan polysulphate seems to be of importance. The release of tissue-type activator plays a decisive role for the regulation of the temporarily insufficient fibrinolytic system, for the thrombolytic process and for the antithrombotic action of pentosan polysulphate.     

A chromogenic assay for the detection of plasmin generated by plasminogen activator immobilized on nitrocellulose using a para-nitroanilide synthetic peptide substrate

A direct solid phase chromogenic assay has been developed for the detection of plasmin (EC 3.4.21.7), generated by the interaction of a nitrocellulose-bound plasminogen activator, using the plasmin specific tripeptide substrate, H-D-valyl-leucyl-lysine - p-nitroaniline. para-Nitroaniline released by the cleavage of the lysine - p-nitroaniline bound by plasmin was derivatized to its diazonium salt and subsequently coupled to N-1-napthylethylenediamine in situ to form a diazoamino of an intense red color at the site of the plasminogen activator. This method was used to assay for the streptococcal plasminogen activator, streptokinase, not only in crude bacterial supernatants, but also to detect streptokinase secreted by individual bacterial colonies. In addition, this solid phase assay was used to identify monoclonal antibodies specific for streptokinase which could inhibit the activation of human plasminogen by streptokinase. This method also permitted simultaneous immunological and biochemical identification of the plasminogen activator, thus permitting unequivocal comparative observations. This assay is quantitative and sensitive to nanogram amounts of activator comparable to those obtained with soluble assays. This method may also be applicable for the detection of other plasminogen activators, such as tissue plasminogen activator, urokinase, and staphylokinase, and also for the detection of immobilized proteases which can cleave other substrates derivatized with p-nitroaniline. The reagents used in this assay are inexpensive and easy to prepare.     

Modulation of plasminogen activator production by interleukin 1: differential responses of fibroblasts derived from human skin and rheumatoid and non-rheumatoid synovium

Rheumatoid synovial fibroblasts were treated with purified porcine interleukin 1 alpha and recombinant human interleukin 1B, and the production of secreted and cell-associated plasminogen activator activity was measured. No stimulation of plasminogen activator activity was seen in response to either preparation of interleukin 1, and in more than half of the cell cultures interleukin 1 caused a significant decrease in the secreted levels of PA activity. Increased levels of prostaglandin E were produced in the same experiments, indicating that the cells were responsive to the interleukin 1 preparations. Both retinoic acid and unfractionated monocyte conditioned medium were able to stimulate the production of PA activity by the rheumatoid synovial fibroblast cultures. The rheumatoid synovial fibroblasts produced two species of plasminogen activator as indicated by SDS polyacrylamide gel electrophoresis, with apparent Mr of approx. 50,000 and 100,000. The Mr = 50,000 species co-migrates with urokinase-type plasminogen activator. No species is produced which co-migrates with tissue type plasminogen activator. Studies with antibodies also indicate that the activity produced is urokinase-type plasminogen activator. The Mr = 100,000 species may be an enzyme-inhibitor complex. Two non-rheumatoid synovial fibroblast cultures and two out of six human skin fibroblast cultures did produce elevated levels of plasminogen activator activity in response to recombinant human interleukin 1B. The results suggest that fibroblast populations may differ in their response to interleukin 1, in terms of production of plasminogen activator activity.     

Decreased plasminogen activator inhibitor-1 secretion in hypoxic corneal epithelial cells is associated with increased urokinase plasminogen activator activity

The aim of this study was to determine the effects of hypoxia on mRNA levels, cell-associated and -secreted protein concentration, activity, and protein complex formation of urokinase-type plasminogen activator, its receptor, and plasminogen activator inhibitor type-1 in corneal epithelium. Non-transformed human corneal epithelial cells were cultured in 20% oxygen (normoxic conditions) or 2% oxygen (hypoxic conditions) for 1, 3, 5, or 7 days. Relative changes in mRNA levels of plasminogen activator, receptor, and plasminogen activator inhibitor-1 were determined using a cDNA expression array, chemiluminescence, and densitometry. Protein concentrations were determined using enzyme linked immunosorbent assays. Activity assays were also used. Protein complex formation was assayed using cell surface biotinylation, immunoprecipitation, and Western blot analysis. Hypoxic corneal epithelial cells demonstrated no significant differences in plasminogen activator or receptor mRNA. Cell-associated plasminogen activator and membrane-associated receptor protein levels were unchanged. In contrast decreases in mRNA and secreted plasminogen activator inhibitor-1 protein were observed in hypoxic cells. Concurrently, increased cell-associated plasminogen activator activity was observed in hypoxic cells. The formation of plasminogen activator/receptor/plasminogen activator inhibitor-1 complex at the cell surface was not inhibited by hypoxia. However, in hypoxic cells less plasminogen activator inhibitor-1 was associated with receptor. It is concluded that in corneal epithelium cultured in 2% oxygen plasminogen activator inhibitor-1 may be an important regulatory factor of the plasminogen activator system resulting in increased urokinase plasminogen activator activity.     

Construction and characterization of a recombinant chimeric plasminogen activator consisting of a fibrin peptide and a low molecular mass single-chain urokinase

A recombinant chimeric plasminogen activator (f beta/scuPA-32k), with a fibrin beta-chain peptide (comprising Gly15 through Arg 42) linked to the N-terminal of a low molecular mass (32 kDa) single-chain urokinase (scuPA-32k, comprising Leu144 through Leu 411) via a 50 amino acid linker sequence, was produced by expression the corresponding chimeric cDNA in Escherichia coli cells. After refolding in vitro, the chimeric protein was purified to homogeneity by zinc chelate-Sepharose chromatography, Sephacryl S200 chromatography and benzamidine-Sepharose chromatography in sequence. The apparent molecular mass was 36 kDa shown by SDS-PAGE analysis. The special activity was 87,000 IU/mg detected by fibrin plate determination. F beta/scuPA-32k could directly activate plasminogen following Michaelis-Menten kinetics with K(m) = 0.52 microM and k(2) = 0.0024 s(-1). Mediated by plasmin, the single-chain molecule could be converted to the active two-chain molecule. The chimeric protein had 3.3 times higher fibrin affinity than scuPA-32k in the fibrin concentration of 3.2 mg/mL, while the chimeric protein inhibited the fibrin clotting and platelet aggregation. F beta/scuPA-32k showed a higher thrombolytic potency in vitro plasma clot lysis than scuPA-32k and depleted less fibrinogen in plasma. These results showed that the chimeric protein had not only higher fibrinolytic activity but also anti-thrombus activity. Further evaluation of the thrombolytic potential in appropriate animal models is required.     

Generation of Novel Chimeric Mice with Humanized Livers by Using Hemizygous cDNA-uPA/SCID Mice

We have used homozygous albumin enhancer/promoter-driven urokinase-type plasminogen activator/severe combined immunodeficient (uPA/SCID) mice as hosts for chimeric mice with humanized livers. However, uPA/SCID mice show four disadvantages: the human hepatocytes (h-heps) replacement index in mouse liver is decreased due to deletion of uPA transgene by homologous recombination, kidney disorders are likely to develop, body size is small, and hemizygotes cannot be used as hosts as more frequent homologous recombination than homozygotes. To solve these disadvantages, we have established a novel host strain that has a transgene containing albumin promoter/enhancer and urokinase-type plasminogen activator cDNA and has a SCID background (cDNA-uPA/SCID). We applied the embryonic stem cell technique to simultaneously generate a number of transgenic lines, and found the line with the most appropriate levels of uPA expression—not detrimental but with a sufficiently damaged liver. We transplanted h-heps into homozygous and hemizygous cDNA-uPA/SCID mice via the spleen, and monitored their human albumin (h-alb) levels and body weight. Blood h-alb levels and body weight gradually increased in the hemizygous cDNA-uPA/SCID mice and were maintained until they were approximately 30 weeks old. By contrast, blood h-alb levels and body weight in uPA/SCID chimeric mice decreased from 16 weeks of age onwards. A similar decrease in body weight was observed in the homozygous cDNA-uPA/SCID genotype, but h-alb levels were maintained until they were approximately 30 weeks old. Microarray analyses revealed identical h-heps gene expression profiles in homozygous and hemizygous cDNA-uPA/SCID mice were identical to that observed in the uPA/SCID mice. Furthermore, like uPA/SCID chimeric mice, homozygous and hemizygous cDNA-uPA/SCID chimeric mice were successfully infected with hepatitis B virus and C virus. These results indicate that hemizygous cDNA-uPA/SCID mice may be novel and useful hosts for producing chimeric mice for use in future long-term studies, including hepatitis virus infection analysis or drug toxicity studies.     

Regulation of urokinase/urokinase receptor interaction by heparin-like glycosaminoglycans

We show here that the interaction between the urokinase-type plasminogen activator and its receptor, which plays a critical role in cell invasion, is regulated by heparan sulfate present on the cell surface and in the extracellular matrix. Heparan sulfate oligomers showing a composition close to the dimeric repeats of heparin (glucosamine-NSO(3)(6-OSO(3))-iduronic acid(2-OSO(3))) n = 5 and n > 5, where iduronic acid may alternate with glucuronic acid, exhibit affinity for urokinase plasminogen activator and confer specificity on urokinase/urokinase receptor interaction. Cell surface clearance of heparan sulfate reduces the affinity of such interaction with a parallel decrease of specific urokinase binding in the presence of an unaltered expression of receptor. Transfection of human urokinase plasminogen activator receptor in normal Chinese hamster ovary fibroblasts and in Chinese hamster ovary cells defective for the synthesis of sulfated glycosaminoglycans results in specific urokinase/receptor interaction only in nondefective cells. Heparan sulfate/urokinase and receptor/urokinase interactions exhibit similar K(d) values. We concluded that heparan sulfate functions as an adaptor molecule that confers specificity on urokinase/receptor binding.