Plasminogen Activator In Endothelial Cells Research Articles

Preliminary findings on the expression of plasma CD63, CD62P, and PAI 1 in patients with acute cerebral infarction

To investigate the expression and clinical significance of lysosomal granule glycoprotein 63 (CD63), P-selectin (CD62P) and endothelial cell plasminogen activator inhibitor (PAI-1) in patients with acute cerebral infarction. A total of 106 patients with acute cerebral infarction (ACI) admitted to our hospital from January to July in 2017 were selected as the patient group; 80 healthy subjects for physical examination in our hospital were selected as the control group. The expression levels of serum CD63, CD62P, and PAI-1 of the subjects were detected. The levels of CD63, CD62P, and PAI-1 in the serum of the patient group were significantly higher than those in the control group. There was a positive correlation between serum CD63 and CD62P (r = 0.672, P < 0.05) in the patient group. There was a positive correlation between serum CD63 and PAI-1 (r = 0.643, P < 0.05) in the patient group. There was also a positive correlation between serum CD62P and PAI-1 (r = 0.601, P < 0.05) in the patient group. Moreover, in other subtypes of cerebral infarction, the expression of CD63, CD62P, and PAI-1 was significantly higher than that of lacunar infarction. CD63, CD62P, and PAI-1 are highly expressed in peripheral blood mononuclear cells (PBMC) and serum of patients with ACI, which may be closely related to the occurrence and development of patients with ACI. These indices may be used as indicators of clinical diagnosis and prognosis in patients with ACI.

Synthesis of 1,4-Diphenylbutadiene Derivatives: Novel Inducer of Tissue-Type Plasminogen Activator (t-PA) in Cultured Bovine Endothelial Cells

(E,E)-1,4-Diphenylbutadiene derivatives were synthesized by utilizing the Stobbe reaction of dimethyl succinate as a key step. Their stereoisomers were also synthesized stereoselectively by means of the cross-coupling reaction of the vinylstannanes and the vinylbromides, which were obtained from the propiolic acid esters by stereoselective hydrostannation, as a key step. To discover novel stimulators of fibrinolysis in vascular endothelial cells, the synthesized compounds were added to cultured bovine endothelial cells to determine the activity of the plasminogen activator in the conditioned medium. Of the synthesized compounds, three compounds were found to stimulate the activity of the plasminogen activator in endothelial cells. In addtition, these compounds inhibited thrombus formation in a rat model of venous thrombosis.

Post-transcriptional regulation of endothelial cell plasminogen activator inhibitor-1 expression during R. rickettsii infection

Intracellular infection of endothelial cells with Rickettsia rickettsii results in increased steady-state levels of plasminogen activator inhibitor-1 (PAI-1) mRNA. Control mechanisms governing such increased expression in response to this novel stimulus have not been defined. In this study, we compared the stability of PAI-1 mRNA in infected and uninfected endothelial cells (EC) and explored the requirement for de novo host cell protein synthesis in the infection-induced increase of steady-state levels. The half-life of PAI-1 mRNA, which is constitutively expressed in cultured EC, increased from 18 h in uninfected EC to greater than 30 h in EC infected for 24 h, a time point at which increases in steady-state PAI-1 mRNA levels are noted. There was no change in stability of γ-actin due to infection. Nuclear run-on studies revealed no apparent increase in transcription rate at 4, 18 and 24 h. R. rickettsii -induced increase in PAI-1 mRNA was blocked by the eukaryotic protein synthesis inhibitor, cycloheximide, which suggests that this response requires de novo host cell protein synthesis. These results provide evidence that post-transcriptional control mechanisms are operative in the regulation of PAI-1 during R. rickettsii infection.

Increased Vascular Endothelial Growth Factor in Tumor Cells and Increased Production of the Receptor for Urokinase Plasminogen Activator in Endothelial Cells Are Associated with Lymph Node Metastasis in Human Breast Cancer

Increased Vascular Endothelial Growth Factor in Tumor Cells and Increased Production of the Receptor for Urokinase Plasminogen Activator in Endothelial Cells Are Associated with Lymph Node Metastasis in Human Breast Cancer

Enzyme-linked immunoassay for midkine, and its application to evaluation of midkine levels in developing mouse brain and sera from patients with hepatocellular carcinomas.

Midkine (MK) is a growth factor that promotes neurite outgrowth and survival of neurons, and enhances the plasminogen activator in endothelial cells. A highly sensitive enzyme-linked immunoassay for MK was developed, involving affinity-purified anti-MK antibodies, their biotinylated form, and avidin-beta-galactosidase. The amount of bound avidin-beta-galactosidase was determined using a fluorogenic substrate, 4-methylumbelliferyl-beta-D-galactoside. This method allowed the detection of human and mouse MK in the range of 50 pg-10 ng. Pleiotrophin, which is related to MK in its amino acid sequence, did not show any cross reactivity. Employing this method, the MK levels in the developing mouse brain were determined. The MK level was 2 micrograms/g of wet tissue on the 12th day of gestation, and then steadily decreased during embryogenesis and postnatal development to 30 ng/g two months after birth. The assay method can also be applied to serum samples. Although the MK levels in the sera of normal human subjects were low or undetectable, 0.6-8 ng/ml of MK was detected in samples in the majority of cases of hepatocellular carcinomas.

Expression of the urokinase receptor in vascular endothelial cells is stimulated by basic fibroblast growth factor.

Basic fibroblast growth factor, a potent angiogenesis inducer, stimulates urokinase (uPA) production by vascular endothelial cells. In both basic fibroblast growth factor-stimulated and -nonstimulated bovine capillary endothelial and human umbilical vein endothelial cells single-chain uPA binding is mediated by a membrane protein with a Mr of 42,000. Exposure of bovine capillary or endothelial human umbilical vein endothelial cells to pmolar concentrations of basic fibroblast growth factor results in a dose-dependent, protein synthesis-dependent increase in the number of membrane receptors for uPA (19,500-187,000) and in a parallel decrease in their affinity (KD = 0.144-0.790 nM). With both cells, single-chain uPA binding is competed by synthetic peptides whose sequence corresponds to the receptor-binding sequence in the NH2-terminal domain of uPA. Exposure of bovine capillary endothelial cells to transforming growth factor beta 1, which inhibits uPA production and upregulates type 1 plasminogen activator inhibitor, the major endothelial cell plasminogen activator inhibitor, has no effect on uPA receptor levels. These results show that basic fibroblast growth factor, besides stimulating uPA production by vascular endothelial cells, also increases the production of receptors, which modulates their capacity to focalize this enzyme on the cell surface. This effect may be important in the degradative processes that occur during angiogenesis.

Restoration of serine protease-inhibitor interaction by protein engineering.

Tissue-type plasminogen activator (t-PA) catalyzes the rate-limiting step in the fibrinolytic cascade: conversion of plasminogen to plasmin. Plasma contains several inhibitors of t-PA that limit its activity and prevent systemic activation of plasminogen. The most important of these is endothelial cell plasminogen activator inhibitor (PAI-1), a member of the serine protease inhibitor (serpin) gene family. We have previously demonstrated that mutation of arginine 304 of t-PA to a glutamic acid residue drastically reduces the rate of interaction between the enzyme and its suicide substrate, PAI-1, without affecting the reactivity of the enzyme toward its normal substrate, plasminogen (Madison, E. L., Goldsmith, E. J., Gerard, R.D., Gething, M.J., and Sambrook, J.F. (1989) Nature 339, 721-724). We report here the use of protein modeling to design a compensatory mutation in PAI-1 (glutamic acid 350 to arginine) and create a molecule that rapidly inhibits this "serpin-resistant" variant of t-PA.

Urokinase-type plasminogen activator in endothelial cells during acute inflammation of the appendix

Urokinase-type plasminogen activator in endothelial cells during acute inflammation of the appendix

Purification from a human hepatoma cell line of a basic fibroblast growth factor-like molecule that stimulates capillary endothelial cell plasminogen activator production, DNA synthesis, and migration.

A 17,500-dalton protein which stimulates plasminogen activator production in cultured bovine capillary endothelial cells has been purified from a SK-Hep-1 human hepatoma cell lysate by using heparin affinity chromatography and fast protein-liquid ion exchange chromatography. The purified molecule stimulated plasminogen activator production in a dose-dependent manner between 0.01 and 1 ng/ml. It also stimulated collagenase synthesis, DNA synthesis, and motility in capillary endothelial cells in the same concentration range. This molecule was identified as a basic fibroblast growth factor-like molecule on the basis of its biological activity, its affinity for heparin-Sepharose, and its cross-reactivity with a polyclonal antibody raised against the human placental basic fibroblast growth factor.

Purification from a human hepatoma cell line of a basic fibroblast growth factor-like molecule that stimulates capillary endothelial cell plasminogen activator production, DNA synthesis, and migration.

A 17,500-dalton protein which stimulates plasminogen activator production in cultured bovine capillary endothelial cells has been purified from a SK-Hep-1 human hepatoma cell lysate by using heparin affinity chromatography and fast protein-liquid ion exchange chromatography. The purified molecule stimulated plasminogen activator production in a dose-dependent manner between 0.01 and 1 ng/ml. It also stimulated collagenase synthesis, DNA synthesis, and motility in capillary endothelial cells in the same concentration range. This molecule was identified as a basic fibroblast growth factor-like molecule on the basis of its biological activity, its affinity for heparin-Sepharose, and its cross-reactivity with a polyclonal antibody raised against the human placental basic fibroblast growth factor.

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.

植物ステロールによる線溶系のこう進

The endothelial cell is a rich source of plasminogen activator that is associated with fibrinolytic activity in blood vessel. Addition of fucosterol or sitosterol to the culture medium of endothelial cells from bovine carotid artery gave rise to a marked increment in the intracellular and extracellular activities of plasminogen activator. Removal of fucosterol or sitosterol from the culture medium resulted in a decrease of plasminogen activator activity back to normal levels. No enhancement of plasminogen activator activity in cultured endothelial cells was observed by cholesterol, 5-androsten-3β-ol and other steroids and sex hormones including androsterone, testosterone, estrone and estradiol. Synthesis of plasminogen activator in cells induced with fucosterol or sitosterol was inhibited by protein synthesis inhibitor, cycloheximide. The plasminogen activators produced in cells were classified into urokinase-type activators with molecular weights of 31, 000 and 55, 000 and tissue-type ones with molecular weights of 81, 000 and 130, 000, which were identified with respective antibodies. The synthesis of each type of plasminogen activator in endothelial cells was stimulated by fucosterol or by sitosterol.

Successive study on the production of plasminogen activator in cultured endothelial cells by phytosterol

In a previous study(1), it was demonstrated that one of phytosterols, sitosterol, has an ability to increase the intracellular and extracellular activities of plasminogen activator in cultured endothelial cells and that other steroids including cholesterol, 5-androsten-3β-ol, stigmasterol, 20(R)propyl-5-pregnen-3β-ol and 20(R)-heptyl-5-pregnen-3β-ol have no ability. Once-stimulated cells went back to normal states by removal of sitosterol. The similar lines of research for plasminogen activators were reported by several groups which were cited in a previous paper(1). In the present communication, we found that fucosterol, which is present mainly in brown algae, Phaeophyta , enhances the production of plasminogen activator in endothelial cells, as well as sitosterol. A similar enhancement was not observed for other steroids and sex hormones including androsterone, testosterone, estrone and estradiol. Synthesis of plasminogen activator induced with fucosterol or sitosterol was inhibited by protein synthesis inhibitor, cycloheximide. The plasminogen activators produced in cells were, in the present study, classified into urokinase-type activators with molecular weights of 31, 000 and 55, 000 and tissue-type ones with molecular weights of 81, 000 and 130, 000, which were identified with respective antibodies. The sy nthesis of each type of plasminogen activator in endothelial cells was stimulated by sitosterol or by fucosterol.

Comparative studies of the fibrinolytic activity of cultured vascular cells

The fibrinolytic components in cultured endothelial cells (ECs) were examined and compared to those in non-endothelial vascular cells (NEVCs) and in normal and virally-transformed fibroblasts. All vascular cells exhibited plasminogen activator (PA) activity. This activity was low in ECs isolated from rabbit aorta and vena cava but high in NEVCs isolated from these same tissues, indicating that the fibrinolytic potential of the vasculature is not restricted to the endothelium. Low PA activity was also characteristic of ECs obtained from human umbilical cord vein. In contrast, both ECs and NEVCs isolated from bovine aorta and vena cava had high PA activity and were even more active in this respect than chick embryo fibroblast transformed by Rous sarcoma virus. Treatment of all cells with 5 × 10 −8 M phorbol myristate acetate, a tumor promoting agent, resulted in a variable but significant stimulation of PA activity. The low PA activity associated with some of the EC cultures correlated with the presence of a cellular inhibitor of fibrinolysis. Inhibitor activity was high in rabbit and human ECs and low in rabbit NEVCs and in chick embryo fibroblasts. No inhibitor activity could be detected in cultures of confluent bovine aortic ECs although there was significant inhibitor activity associated with sparsely populated cultures. The specific activity of PA in ECs also fluctuated with cell density, increasing approximately 20-fold as sparsely plated cells grew to confluency. These observations emphasize 1) that tissue associated fibrinolytic activity represents a balance between the activities of PAs and cellular inhibitors, 2) that the relation between PA and inhibitor may change with the growth state of the cells, and 3) that these activities vary with species of origin and thus are not necessarily indicative of any specific cell type.