The balance between tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor type 1 (PAI-1) regulates fibrinolysis. PAI-1 expression increases in atherosclerotic arteries and vascular smooth muscle cells (VSMCs) are one of major constituents of atheroma. We investigated the impact of lysophosphatidylcholine (lysoPC), an active component of oxidized low-density lipoprotein, on the plasminogen activator system of the rat VSMCs. The lysoPC stimulated the protein and gene expressions of PAI-1 but did not affect the protein expression of t-PA. Fibrin overlay zymography revealed that lysoPC increased the activity of PAI-1 in the conditioned media, while concurrently decreasing that of free t-PA. Vitamin E inhibited the lysoPC-induced PAI-1 expression. Further, lysoPC increased the intracellular reactive oxygen species (ROS) formation. Caffeic acid phenethyl ester, an inhibitor of NF-kappaB, blocked this lysoPC effect. Indeed, lysoPC induced the NF-kappaB-mediated transcriptional activity as measured by luciferase reporter assay. In addition, genistein, an inhibitor of protein-tyrosine kinase (PTK), diminished the lysoPC effect, while 7,12-dimethylbenz[a]anthracene, a stimulator of PTK, stimulated PAI-1 production. In conclusion, lysoPC does not affect t-PA expression but induces PAI-1 expression in the VSMC by mediating NF-kappaB and the genistein-sensitive PTK signaling pathways via oxidative stress. Importantly, lysoPC stimulates the enzyme activity of PAI-1 and suppresses that of t-PA.
Animals ; Benz(a)Anthracenes/pharmacology ; Caffeic Acids/pharmacology ; Cells, Cultured ; Genistein/pharmacology ; Lipoproteins, LDL/metabolism ; Lysophosphatidylcholines/*pharmacology ; Muscle, Smooth, Vascular/cytology/*drug effects/metabolism ; NF-kappa B/antagonists & inhibitors/metabolism ; Oxidative Stress/drug effects ; Phenylethyl Alcohol/analogs & derivatives/pharmacology ; Plasminogen Activator Inhibitor 1/agonists/genetics/*metabolism ; Protein Kinase Inhibitors/pharmacology ; Protein-Tyrosine Kinases/antagonists & inhibitors/metabolism ; Rats ; Rats, Sprague-Dawley ; Reactive Oxygen Species/metabolism ; Signal Transduction/drug effects ; Tissue Plasminogen Activator/*metabolism ; Transcription, Genetic/drug effects ; Up-Regulation/drug effects ; Vitamin E/pharmacology

Toxoplasma gondii can modulate host cell gene expression; however, determining gene expression levels in intermediate hosts after T. gondii infection is not known much. We selected 5 genes (ALDH1A2, BEX2, CCL3, EGR2 and PLAU) and compared the mRNA expression levels in the spleen, liver, lung and small intestine of genetically different mice infected with T. gondii. ALDH1A2 mRNA expressions of both mouse strains were markedly increased at day 1-4 postinfection (PI) and then decreased, and its expressions in the spleen and lung were significantly higher in C57BL/6 mice than those of BALB/c mice. BEX2 and CCR3 mRNA expressions of both mouse strains were significantly increased from day 7 PI and peaked at day 15-30 PI (P<0.05), especially high in the spleen liver or small intestine of C57BL/6 mice. EGR2 and PLAU mRNA expressions of both mouse strains were significantly increased after infection, especially high in the spleen and liver. However, their expression patterns were varied depending on the tissue and mouse strain. Taken together, T. gondii-susceptible C57BL/6 mice expressed higher levels of these 5 genes than did T. gondii-resistant BALB/c mice, particularly in the spleen and liver. And ALDH1A2 and PLAU expressions were increased acutely, whereas BEX2, CCL3 and EGR2 expressions were increased lately. Thus, these demonstrate that host genetic factors exert a strong impact on the expression of these 5 genes and their expression patterns were varied depending on the gene or tissue.
Aldehyde Dehydrogenase/*genetics/metabolism ; Animals ; Brain/metabolism/parasitology ; Chemokine CCL3/*genetics/metabolism ; Early Growth Response Protein 2/*genetics/metabolism ; Gene Expression Profiling ; Humans ; Lung/metabolism/parasitology ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Mice, Inbred Strains ; Nerve Tissue Proteins/*genetics/metabolism ; Organ Specificity ; Spleen/metabolism/virology ; Toxoplasma/*physiology ; Toxoplasmosis/*genetics/metabolism/parasitology ; Urokinase-Type Plasminogen Activator/*genetics/metabolism

BACKGROUNDCigarette smoking has an influence on both arterial-type and venous-type thrombosis. However, little is known about the direct effect of cigarette smoke extract (CSE) on fibrinolytic activity of human umbilical vein endothelial cells (HUVECs). Most recently, simvastatin has been marked in its effect on endothelial cells protection and anticoagulation. In this study, the effect of CSE on the expression of tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) in HUVECs was addressed. The role of simvastatin in CSE-induced fibrinolytic activity changes was investigated as well.

METHODSThe fourth to fifth generation of HUVECs were incubated respectively with 0, 5%, 10% and 20% CSE for 6 hours or exposed to 5% CSE for 0, 4, 6, 8, 12, 24 hours to determine the expression changes of t-PA and PAI-1 protein. Meanwhile, cells were also accordingly exposed either to 5% CSE alone or simvastatin pre-treated and 5% CSE for 24 hours to assess the role of simvastatin in CSE-induced t-PA and PAI-1 protein and mRNA expression in HUVECs. RT-PCR and ELISA techniques were used for detecting the t-PA or PAI-1 mRNA and protein.

RESULTSAfter 6-hour exposure to CSE, the expression levels of t-PA protein in 10% and 20% CSE-treated groups reduced significantly ((0.0365 +/- 0.0083) ng/ml, (0.0255 +/- 0.0087) ng/ml) when compared with that of control group ((0.0660 +/- 0.0120) ng/ml) (P < 0.05). In contrast, the levels of PAI-1 protein in 5%, 10% and 20% CSE-treated groups increased remarkably ((13.3225 +/- 0.5680) ng/ml, (14.2675 +/- 1.5380) ng/ml, (14.4292 +/- 1.6230) ng/ml) when compared with that of control group ((8.5193 +/- 0.7537) ng/ml) (P < 0.05). After stimulation with 5% CSE for 0, 4, 6, 8, 12, 24 hours, the levels of PAI-1 protein increased over time and reached the peak at 24 hours ((14.6400 +/- 1.0651) ng/ml), which was significantly higher than that of control group ((12.0656 +/- 0.6148) ng/ml) (P < 0.05). Additionally, CSE could up-regulate PAI-1 expression at both the mRNA and the protein levels. The levels of PAI-1 mRNA and protein increased significantly in 5% CSE-treated group ((8.8030 +/- 0.4745) ng/ml, (1.8155 +/- 0.0412) ng/ml) compared with those of control groups ((5.0588 +/- 0.2315) ng/ml, (1.3030 +/- 0.0647) ng/ml) (P < 0.01), and decreased after 2-hour simvastatin pre-treatment ((5.4875 +/- 0.3166) ng/ml, (1.3975 +/- 0.0297) ng/ml) (P < 0.01). No significant difference was found at the levels of t-PA protein and mRNA (P > 0.05).

CONCLUSIONSCSE inhibits the fibrinolytic activity of HUVECs in vitro. Simvastatin plays a protective role in CSE-induced fibrinolytic malfunction.

Cells, Cultured ; Endothelial Cells ; metabolism ; Fibrinolysis ; drug effects ; Humans ; Hydroxymethylglutaryl-CoA Reductase Inhibitors ; pharmacology ; Plasminogen Activator Inhibitor 1 ; analysis ; biosynthesis ; genetics ; Simvastatin ; pharmacology ; Smoke ; adverse effects ; Tissue Plasminogen Activator ; analysis ; biosynthesis ; genetics ; Tobacco ; adverse effects ; Umbilical Veins ; cytology

OBJECTIVETo investigate the effect of urokinase on hepatic fibrogenesis in rats.

METHODSHepatic fibrosis was induced in rats by complex pathogenic factors including subcutaneous injections of carbon tetrachloride, alcohol and cholesterol feeding. Animals were randomly divided into 3 groups: normal control group, hepatic fibrosis group (complex pathogenic factors for 6 weeks), UK prevention group (complex pathogenic factors+UK for 6 weeks). The animals were sacrificed at the end of week 6. The expression of alpha-SMA, uPA, PAI-1, TGFb1, TIMP-1, collagen type I and type III proteins in hepatic fibrosis tissue was detected by immunohistochemistry, the expression of PAI-1 and TGFb1 mRNA in the hepatic fibrosis tissue was quantified by real time RT-PCR. The serum levels of hyaluronicacid (HA), alanine aminotransferase (ALT), aspartate aminotransferase (AST), bilirubin (TBil) and the content of liver hydroxyproline (Hyp) were detected using ELISA kits.

RESULTSThe serum ALT, AST, TBil, HA and the content of liver Hyp were (46.66+/-6.30) U/L, (126.26+/-31.65) U/L, (31.11+/-4.20) micromol/L, (109.70+/-18.81) microg/L and (0.98+/-0.09) mg/(g liver), respectively, in UK prevention group, which were significantly lower than those [(101.57+/-11.97) U/L, (205.89+/-56.26) U/L, (67.75+/-2.75) micromol/L, (184.43+/-32.36) microg/L and (1.65+/-0.16) mg/(g liver), respectively] in hepatic fibrosis group (q = 3.3801-20.0061, P < 0.01). The levels of a-SMA, collagen type I, type III, TIMP-1, PAI-1, TGFb1 proteins were (299.27+/-37.36), (210.05+/-27.17), (192.94+/-24.48), (213.70+/-32.21), (204.25+/-17.92), (205.97+/-23.81), respectively, in UK prevention group, which were significantly lower than those [(418.83+/-30.21), (323.77+/-21.53), (302.37+/-31.43), (376.63+/-25.19), (313.53+/-26.67) and (327.42+/-36.75), respectively] in hepatic fibrosis group. The level of uPA protein was increased, and the expression of PAI-1, TGFb1 mRNA in hepatic fibrosis tissue was decreased in UK prevention group.

CONCLUSIONIn the early stage of hepatic fibrogenesis, urokinase can attenuate the progression of rat hepatic fibrosis via upregulation of uPA, downregulation of TGFb1, and inhibition of HSC activation.

Actins ; metabolism ; Animals ; Disease Models, Animal ; Hydroxyproline ; metabolism ; Liver ; drug effects ; metabolism ; pathology ; Liver Cirrhosis, Experimental ; chemically induced ; metabolism ; pathology ; prevention & control ; Liver Function Tests ; Male ; Plasminogen Activator Inhibitor 1 ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Random Allocation ; Rats ; Rats, Wistar ; Tissue Inhibitor of Metalloproteinase-1 ; genetics ; metabolism ; Transforming Growth Factor beta1 ; genetics ; metabolism ; Urokinase-Type Plasminogen Activator ; pharmacology

In this study, we assessed the effects of shear stress on the expression of plasminogen activator(tPA and uPA) mRNA in cultured NRK-52E cells (a kidney proximal tubular epithelial cell line of normal rat origin) and investigated the mechanism of tubulointerstitial extracellular matrix (ECM) remodeling in the early stage of diabetic nephropathy (DN). The cultured NRK-52E cells were exposed to shear stress of 5 and 10 dyn/cm2 for 1, 3 and 6 hours respectively. Semi-quantity RT-PCR was used to detect the expression of tPA and uPA mRNA. Shear stress down-regulated the expression of tPA and uPA mRNA in cultured NRK-52E cells in a magnitude and time-dependent way. The results suggested that the increased tubular shear stress in the early-stage of DN could decrease the expression of tPA and uPA in renal proximal tubular cells, lead to the reduction of tubulointerstitial fibrinolytic activity and involve in the remodeling of ECM.
Animals ; Cell Line ; Diabetic Nephropathies ; pathology ; Epithelial Cells ; cytology ; metabolism ; pathology ; Extracellular Matrix ; metabolism ; Humans ; Kidney Tubules, Proximal ; cytology ; metabolism ; pathology ; RNA, Messenger ; genetics ; metabolism ; Rats ; Shear Strength ; Tissue Plasminogen Activator ; genetics ; metabolism ; Urokinase-Type Plasminogen Activator ; genetics ; metabolism

OBJECTIVETo study the activities of the key enzymes in reteplase production by Pichia pastoris.

METHODSIn shaking flasks, a series of samples were maintained after methanol induction. The cells were sonicated to prepare cell-free suspensions, in which the activities of AOX, FAD, PDC, G-6-PD, ID, alpha-KGD and SD were measured.

RESULTSThe specific activity of AOX increased during the initial 6 h, reaching the maximum of 44.5 U/mg protein. The activity decreased quickly between 6 and 24 h, followed by increment in the following 24 h and decreased afterwards. The specific activity of FAD increased gradually in the initial 48 h and then decreased, with the peak level of 6.72 U/mg protein occurred at 48 h. The specific activity of G-6-PD increased at in 2-6 h and 24-48 h, but decreased in 6-24 h and after 48 h. The specific activity of PDC decreased during the initial 6 h and increased slowed afterwards. The specific activities of ID, alpha-KGD and SD all showed a rapid decrease in the initial 6 h and a slow decrease in 6-24 h. After 24 h, the activity of ID continued to decrease, but the other two increased in the following 24 h and then decreased, reaching the maximum at 48 h.

CONCLUSIONSAccording to the changes of these enzyme activities, the whole induction phase can be divided into 4 stages: the methanol-adaptive period in the initial 6 h, the fast growth period between 6 and 24 h, the product accumulation period in 24-48 h and the metabolism lag period in 48-72 h. In the methanol-adaptive period, complete oxidation of methanol is the dominant pathway. But in the following two stages, the metabolic pathway shifts towards glycolysis and TCA cycle.

Alcohol Oxidoreductases ; metabolism ; Fermentation ; Genetic Vectors ; Glucosephosphate Dehydrogenase ; metabolism ; Methanol ; pharmacology ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; metabolism ; Tissue Plasminogen Activator ; biosynthesis ; genetics ; metabolism

Animals ; Annexin A2 ; chemistry ; genetics ; metabolism ; physiology ; DNA Replication ; Endocytosis ; Exocytosis ; Humans ; Neoplasm Invasiveness ; Neoplasm Metastasis ; Neoplasms ; metabolism ; pathology ; Prognosis ; Tissue Plasminogen Activator ; metabolism ; physiology

We have established a human umbilical vein endothelial cell (HUVEC) line monoclonal cells with the stable expression of human tissue-type plasminogen activator (t-PA) gene to provide a basis for further study on the vascular tissue engineering. Recombinant plasmid pcDNA3. 1-Myc-His B (-)/t-PA was constructed by insertion of t-PAcDNA originated from PBS/t-PA into eukaryotic expression vector pcDNA3. 1-Myc-His B(-) and transfected into hUVEC line cells mediated by lipofectamine. The positive clones were obtained by the screen of G418. The transcription and expression of t-PA gene were investigated by RT-PCR and Western blotting respectively. The t-PA activity was measured by chromogenic substrate assay. The positive clone cells which transcripted the mRNA of t-PA gene was obtained by RT-PCR. Immunoreactive human t-PA of the medium was significantly increased in the group of transfected gene when compared with that in the controlled and transfected plasmid without t-PA gene group. The biological activity of the protein of the t-PA in the media was increased significantly in the positive clone cells with t-PA gene transfected. The HUVEC line monoclonal cells with the stable expression of t-PA gene was established successfully.
Cell Line ; Human Umbilical Vein Endothelial Cells ; metabolism ; Humans ; Lipids ; pharmacology ; RNA, Messenger ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Tissue Engineering ; Tissue Plasminogen Activator ; biosynthesis ; genetics ; Transfection

OBJECTIVETo analyze the phenotypic and functional characteristics of endothelial (T3A) cells derived from human hepatocellular cell carcinoma.

METHODSEndothelial cells were isolated from human hepatocellular carcinoma specimens. The identification of T3A cells was performed by checking von Willebrand Factor (vWF), CD31, CD34 and Dil-Ac-LDL uptake. The cell surface fenestrations, a specific morphological feature of tumor derived EC, were investigated by scanning and transmission electron microscopy. The phenotypic characteristics of T3A cells were analyzed by fluorescence-activated cell sorter (FACS) and were further conformed by real-time PCR at transcription level. Furthermore, tumor necrosis factor alpha (TNFalpha)-induced cytotoxicity was evaluated by 3-(4, 5-dimethythiazolyl) -2, -diphenyl-2H-tetrazolium-bromide (MTT) assay; Matrix metalloproteinase secretion was detected by zymography; Angiogenic ability in vitro was analyzed by culturing T3A cells in three-dimensional Matrigel plug. Coagulant and fibrinolytic activities were detected by enzyme-linked immunosorbent assay (ELISA).

RESULTSThe isolated T3A cells exhibited classic "spindle-shape" morphology and monolayer growth and contact inhibition properties. Immunofluorescent staining showed that T3A cells expressed vWF, CD31, CD34, and uptake of Dil-Ac-LDL at a high level. The cell surface fenestrations were observed on T3A cells by scanning and transmission electron microscopy. By FACS and real-time PCR, T3A cells were found to express alphav3, alphavbeta5 and TNF receptor p75 at high levels, and TNF receptor p55 and ICAM-1 at low levels, as compared with those in human liver sinusoidal endothelial cells (LSEC). In response to TNFalpha, LSEC exhibited a dose-dependent cytotoxicity, while T3A cells were resistant. Gelatin zymography showed that MMP-2 activity was higher in T3A cells than that in LSEC. In a three-dimensional plug of Matrigel, T3A cells exhibited stronger angiogenic ability as compared with LSEC. In addition, T3A cells released more tissue factor (TF), tissue-type plasminogen activator (t-PA), plasminogen activator inhibitor (PAI-1) and urine plasminogen activator (u-PA) than LSEC in response to TNFalpha.

CONCLUSIONTumor-derived endothelial cells are phenotypically and functionally different from those derived from normal liver tissue.

Antigens, CD34 ; metabolism ; Carcinoma, Hepatocellular ; genetics ; metabolism ; pathology ; Cell Proliferation ; drug effects ; Cell Shape ; Cells, Cultured ; Endothelial Cells ; metabolism ; pathology ; ultrastructure ; Gene Expression ; Humans ; Integrin alphaVbeta3 ; metabolism ; Integrins ; metabolism ; Intercellular Adhesion Molecule-1 ; metabolism ; Lipoproteins, LDL ; metabolism ; Liver Neoplasms ; genetics ; metabolism ; pathology ; Lung ; blood supply ; metabolism ; pathology ; Matrix Metalloproteinase 2 ; metabolism ; Microscopy, Electron, Scanning ; Neovascularization, Pathologic ; metabolism ; pathology ; Phenotype ; Plasminogen Activator Inhibitor 1 ; metabolism ; Platelet Endothelial Cell Adhesion Molecule-1 ; metabolism ; Receptors, Tumor Necrosis Factor, Type I ; metabolism ; Receptors, Vitronectin ; metabolism ; Tissue Plasminogen Activator ; metabolism ; Tumor Cells, Cultured ; Tumor Necrosis Factor Decoy Receptors ; metabolism ; Tumor Necrosis Factor-alpha ; pharmacology ; von Willebrand Factor ; metabolism

BACKGROUNDTissue-engineered heart valves have the potential to overcome the limitations of present heart valve replacements. This study was designed to develop a tissue engineering heart valve by using human umbilical cord blood-derived endothelial progenitor cells (EPCs) and decellularized valve scaffolds.

METHODSDecellularized valve scaffolds were prepared from fresh porcine heart valves. EPCs were isolated from fresh human umbilical cord blood by density gradient centrifugation, cultured for 3 weeks in EGM-2-MV medium, by which time the resultant cell population became endothelial in nature, as assessed by immunofluorescent staining. EPC-derived endothelial cells were seeded onto the decellularized scaffold at 3 x 10(6) cells/cm(2) and cultured under static conditions for 7 days. Proliferation of the seeded cells on the scaffolds was detected using the MTT assay. Tissue-engineered heart valves were analyzed by HE staining, immunofluorescent staining and scanning electron microscopy. The anti-thrombogenic function of the endothelium on the engineered heart valves was evaluated by platelet adhesion experiments and reverse transcription-polymerase chain reaction (RT-PCR) analysis for the expression of endothelial nitric oxide synthase (eNOS) and tissue-type plasminogen activator (t-PA).

RESULTSEPC-derived endothelial cells showed a histolytic cobblestone morphology, expressed specific markers of the endothelial cell lineage including von Willebrand factor (vWF) and CD31, bound a human endothelial cell-specific lectin, Ulex Europaeus agglutinin-1 (UEA-1), and took up Dil-labeled low density lipoprotein (Dil-Ac-LDL). After seeding on the decellularized scaffold, the cells showed excellent metabolic activity and proliferation. The cells formed confluent endothelial monolayers atop the decellularized matrix, as assessed by HE staining and immunostaining for vWF and CD31. Scanning electron microscopy demonstrated the occurrence of tight junctions between cells forming the confluent monolayer. Platelets adhesion experiments suggested that the neo-endothelium was non-thrombogenic. The expression levels of eNOS and t-PA genes in the neo-endothelium were quite similar to those in human umbilical vein endothelial cells.

CONCLUSIONSEPCs isolated from the human umbilical cord blood can differentiate into endothelial cells in vitro and form a functional endothelium atop decellularized heart valve scaffolds. Thus, EPCs may be a promising cell source for constructing tissue-engineered heart valves.

Animals ; Cell Proliferation ; Endothelial Cells ; cytology ; metabolism ; Heart Valve Prosthesis ; Heart Valves ; cytology ; metabolism ; ultrastructure ; Humans ; Immunohistochemistry ; Microscopy, Electron, Scanning ; Nitric Oxide Synthase Type III ; genetics ; metabolism ; Platelet Aggregation ; Reverse Transcriptase Polymerase Chain Reaction ; Stem Cells ; cytology ; metabolism ; Swine ; Tissue Engineering ; methods ; Tissue Plasminogen Activator ; genetics ; metabolism ; Umbilical Cord ; cytology