AIMIn order to elucidate the relationship between homocysteine (Hcy) and the fibrinolytic system, we examined the effect of Hcy on tissue- type plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1) gene expression in human umbilical vein endothelial cells (HUVEC) in vitro.

METHODSTotal RNA was extracted from HUVEC exposed to physical and pathological concentrations of Hcy (0, 10, 50, 200, 500 micromol/L ) for 24 hours, using the guanidinium isothiocyanate method. The semi-quantification of tPA and PAI-1 mRNA in HUVEC was carried out by reverse transcriptase-polymerase chain reaction (RT-PCR).

RESULTSPAI-1 mRNA levels was enhanced by Hcy at concentrations of 500 micromol/L of Hcy, compared with that of 0 micromol/L Hcy (P < 0.05). The mRNA expression of tPA, however, was significantly decreased at concentrations of 500 micromol/L Hcy, compared with that of 10 micromol/L Hcy (P < 0.05), but compared with the control group, the tPA level of 10 micromol/L Hcy was much higher (P < 0.05).

CONCLUSIONHyperhomocysteinemia increases the incidence of cardio cerebral vascular disease, which may be caused by decreasing the activity of fibrinolytic system, whereas, the physiological concentration of Hcy may be decreased the incidence by enhancing the activity of fibrinolytic system.

Cells, Cultured ; Gene Expression ; Homocysteine ; administration & dosage ; pharmacology ; Human Umbilical Vein Endothelial Cells ; drug effects ; metabolism ; Humans ; Plasminogen Activator Inhibitor 1 ; genetics ; metabolism ; RNA, Messenger ; genetics ; Tissue Plasminogen Activator ; genetics ; metabolism

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 role of plasminogen activator inhibitor-1 (PAI-1) and tissue plasminogen activator (tPA) in the accumulation of extracellular matrix (ECM) in the kidney of KKAy mice with type 2 diabetes.

METHODSKKAy mice, a type 2 diabetic animal model, and C57BL-J mice were sacrificed at 16, 20, and 24 weeks of age, respectively. The local expression of renal laminin was analyzed with immunohistochemistry. Chromogenic substance was used to show the activity of PAI-1. The mRNA expression of tPA was determined by RT-PCR. The mRNA expression of PAI-1 was measured by reverse transcription-polymerase chain reaction (RT-PCR).

RESULTSLaminnin expression was significantly increased in all age groups of KKAy mice. The tPA mRNA was significantly lower than that in C57BL mice, especially at the age of 16w (only 47%). Otherwise the PAI-1 mRNA expression was remarkably up-regulated than that in C57BL mice.

CONCLUSIONIn type 2 diabetes KKAy mice, the accumulation of ECM may be associated with the abnormal expression of PAI-1/tPA mRNA.

Animals ; Diabetes Mellitus, Experimental ; metabolism ; Diabetes Mellitus, Type 2 ; metabolism ; Extracellular Matrix ; metabolism ; Kidney ; metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Plasminogen Activator Inhibitor 1 ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Tissue Plasminogen Activator ; biosynthesis ; genetics

Actins ; biosynthesis ; genetics ; Adult ; Aged ; Female ; Hepatitis B, Chronic ; complications ; Humans ; Liver Cirrhosis ; blood ; enzymology ; virology ; Male ; Matrix Metalloproteinase 1 ; biosynthesis ; genetics ; Middle Aged ; Plasminogen Activator Inhibitor 1 ; blood ; Tissue Inhibitor of Metalloproteinase-1 ; biosynthesis ; genetics ; Urokinase-Type Plasminogen Activator ; blood

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

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

To combine the fibrinolytic with anticoagulant activities for therapy of thrombotic deseases, a fusion protein made of tissue-type plasminogen activator (t-PA) and hirudin was constructed and expressed in chia pastoris. To improve thrombolytic properties of t-PA and reduce bleeding side effect of hirudin, FXa-recognition sequence was introduced between t-PA and hirudin molecules.The anticoagulant activity of hirudin can be target-released through cleavage of FXa at thrombus site. t-PA gene and hirudin gene with FXa-recognition sequence at its 5'-terminal were obtained by RT-PCR and PCR respectively. The fusion protein gene was cloned into plasmid pIC9K and electroporated into the genome of Pichia pastoris GS115. The expression of fusion protein was induced by methanol in shaking flask and secreted into the culture medium. Two forms of the fusion protein, single-chain and double-chain linked by a disulfide bond (due to the cleveage of t-PA at Arg275-Ile276), were obtained. The intact fusion protein retained the fibrinolytic activity but lacked any anticoagulant activity. After cleavage by FXa, the fusion protein liberated intact free hirudin to exert its anticoagulant activity. So, the fusion protein is a bifunctional molecule having good prospect to develop into a new targeted therapeutic agent with reduced bleeding side effect for thrombotic diseases.
Animals ; Cloning, Molecular ; Electroporation ; Hirudins ; biosynthesis ; genetics ; Humans ; Pichia ; genetics ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Tissue Plasminogen Activator ; biosynthesis ; genetics

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

OBJECTIVETo detect the expressing levels of human tissue-plasminogen activator(t-PA) in AGZY83-a cells transfected with pcDNA3.1(+) t-PA in vivo and the feasibility of using transplantation of cells for gene therapy of thrombotic diseases.

METHODSExpression vectors containing the t-PA cDNA gene were transfected into AGZY83-a cells. The transfected AGZY83-a cells were implanted into mice in different regions, and the plasma levels of human t-PA were assayed at intervals.

RESULTSThe plasma levels of human t-PA were significantly increased in mice after implantation of transfected AGZY83-a cells and were significantly higher than those of control groups implanted with untransfected AGZY83-a cells. This significant increase lasted at least 105 days. The intraperitoneal implantation group expressed the highest level of human t-PA, a little higher than that of the subcutis implantation group, while both are much higher than that of the quadriceps femoris implantation group which expressed the lowest.

CONCLUSIONThe implanted transfected AGZY83-a cells are able to stably express high levels of human t-PA, and transplantation of cells transfected with pcDNA3.1(+) t-PA is a new promising method for gene therapy of thrombotic diseases.

Animals ; Cell Line ; Cell Transplantation ; methods ; Gene Expression ; Humans ; Mice ; Plasmids ; genetics ; Time Factors ; Tissue Plasminogen Activator ; blood ; genetics ; Transfection ; Transplantation, Heterologous

Tissue plasminogen activator (t-PA) and plasminogen activator inhibitor type 1 (PAI-1) may be involved in the pathogenesis of peptic ulcers through suppression of fibrinolysis. This study was designed to investigate associations of t-PA and PAI-1 genes with clinical features of the patients with bleeding gastric ulcers. Eighty-four patients with peptic ulcers and 100 controls were studied between January 1998 and April 2000. We used polymerase chain reaction and endonuclease digestion to genotype for 4G/5G polymorphism in the promoter region of the PAI-1 gene and the Alurepeat insertion/deletion (I/D) polymorphism in intron h of the t-PA gene. Various clinical features, including lesion site, bleeding event, recurrence of ulcer, and rebleeding, were assessed using a multiple logistic regression model. The genotype distributions of both the t-PA and PAI-1 genes did not differ between the patient and control groups. The occurrence of the I/D or D/D genotype of t-PA was significantly higher in cases of duodenal ulcer (adjusted OR=4.39, 95% CI=1.12-17.21). When a dominant effect (i.e., 4G/4G or 4G/5G versus 5G/5G) of the 4G allele was assumed, the PAI-1 4G/4G genotype was independently associated with rebleeding after hemostasis (adjusted OR=5.07, 95% CI=1.03-24.87). Our data suggest that t-PA gene polymorphism is associated with duodenal ulcers, and that the PAI-1 gene may be a risk factor leading to recurrent bleeding after initial hemostasis.
Adult ; Aged ; Alu Elements/genetics ; DNA Mutational Analysis ; Duodenal Ulcer/complications ; Duodenal Ulcer/genetics* ; Female ; Gene Frequency ; Genetic Predisposition to Disease ; Genotype ; Human ; Male ; Middle Aged ; Mutagenesis, Insertional ; Peptic Ulcer Hemorrhage/etiology ; Peptic Ulcer Hemorrhage/genetics* ; Plasminogen Activator Inhibitor 1/genetics* ; Polymorphism (Genetics)* ; Promoter Regions (Genetics)/genetics ; Recurrence ; Sequence Deletion ; Stomach Ulcer/complications ; Stomach Ulcer/genetics* ; Tissue Plasminogen Activator/genetics*