Adenoviridae ; genetics ; Gene Expression ; Hepatic Stellate Cells ; metabolism ; Humans ; Urokinase-Type Plasminogen Activator ; genetics ; metabolism

OBJECTIVETo construct the expression vector of human tissue factor (TF), and investigate the influence of TF/coagulant factor VIIa (FVIIa) complex on the transcriptional expression of urokinase plasminogen activator (u-PA) and u-PA receptor (u-PAR) in human ovarian cancer.

METHODSThe human TF cDNA was obtained from placenta by RT-PCR and then inserted into eukaryotic expression vector pcDNA3 to obtain the TF-pcDNA3 combinant. This combinant was transfected into human ovarian cancer cell line A2780 by lipofectamine. Stably-transfected cells A2780/TF were screened. A2780 and A2780/TF cell lines were stimulated by FVIIa respectively, and the transcriptional levels of u-PA and u-PAR were examined by RT-PCR.

RESULTS(1) The constructed product was identified as TF-pcDNA3 combinant by sequencing. (2) TF was highly expressed not only at transcriptional level in the stable-transfected A2780/TF cell (transfected cell 3.91 +/- 0.28, untransfected cell 0.97 +/- 0.23, P < 0.01), but also on the membrane of the cell surface [transfected cell (48.56 +/- 9.53)%, untransfected cell (2.73 +/- 1.15)%, P < 0.01]. (3) The u-PA and u-PAR mRNA levels in A2780 cell line did not change significantly after stimulated by FVIIa; (4) While stimulated by FVIIa, the u-PAR mRNA levels in A2780/TF cells increased significantly in both dose-dependent and time-dependent manner, while the u-PA mRNA levels did not change significantly; (5) In the A2780/TF cell line the enhanced expression of u-PAR mRNA by FVIIa was significantly inhibited by coincubated with anti-TF antibody.

CONCLUSIONTF/FVIIa complex could up-regulate the transcription of u-PAR in human ovarian cancer cells so as to enhance tumor invasion and metastasis.

Cell Line, Tumor ; Factor VIIa ; genetics ; metabolism ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Ovarian Neoplasms ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Receptors, Urokinase Plasminogen Activator ; genetics ; metabolism ; Thromboplastin ; genetics ; metabolism ; Up-Regulation ; Urokinase-Type Plasminogen Activator ; genetics ; metabolism

AIMTo investigate the gene expression changes of urokinase plasminogen activator (uPA)/urokinase receptor (uPAR) in rat testes at postnatal stages and explore the effects of uPA/uPAR system on the rat spermatogenesis.

METHODSThe mRNAs of uPA and uPAR in rat testes were measured by using real-time quantitative polymerase chain reaction (PCR) at postnatal days 0, 5, 10, 15, 21, 28, 35, 42, 49 and 56, respectively.

RESULTSThe tendencies of uPA and uPAR mRNA expression were similar at most postnatal stages except for D(0). The expression of uPAR mRNA in rats testes was relatively higher than that of uPA at postnatal D(0), and both were decreased until D(21), increased obviously at postnatal D(28), reached a peak at postnatal D(35), then declined sharply at postnatal D(42) and retained at a low level afterwards.

CONCLUSIONThe uPA/uPAR system may be strongly linked to spermiation and spermatogenesis via regulating germ cell migration and proliferation, as well as promoting the spermiation and detached residual bodies from the mature spermatids.

Aging ; genetics ; Animals ; Animals, Newborn ; Gene Expression Regulation, Developmental ; Gene Expression Regulation, Enzymologic ; Male ; Polymerase Chain Reaction ; Rats ; Receptors, Cell Surface ; genetics ; Receptors, Urokinase Plasminogen Activator ; Spermatogenesis ; Spermatozoa ; enzymology ; physiology ; Testis ; growth & development ; physiology ; Urokinase-Type Plasminogen Activator ; genetics

The fusion protein of Humanized mouse anti-human fibrin ScFv and the low molecular weight urokinase (IIn-UK) contained seven disulfide bonds and formed inclusion body while expressing in normal E. coli strain. By coexpressing DsbC and using the special E. coli strain Origami(DE3) which was trxB/gor double mutant, the fusion protein IIn-UK was functionally expressed in the cytoplasm of E. coli. The expressed fusion protein in the soluble fraction was purified by using affinity chromatography specific against urokinase. The purified fusion protein could combine the thrombus in vitro, and the specific activity of urokinase reached 80,000 IU/mg fusion protein. The result showed that the fusion protein retained the activity of two moieties, and this study laid a foundation for further research of targeting thrombolytic agent.
Animals ; Chromatography, Affinity ; Escherichia coli ; genetics ; Gene Expression ; Humans ; Immunoglobulin Fragments ; genetics ; Mice ; Recombinant Fusion Proteins ; genetics ; isolation & purification ; metabolism ; Urokinase-Type Plasminogen Activator ; genetics

BACKGROUNDRecent studies have proved that brain-derived neurotrophic factor (BDNF) possesses angiogenic activity in vitro and in vivo. However, the proangiogenic mechanism of BDNF has not yet been provided with enough information. To explore the proangiogenic mechanism of BDNF, we investigated the effects of BDNF on extracellular proteolytic enzymes, including matrix metalloproteinases (MMPs) and serine proteases, particularly the urokinase-type plasminogen activator (uPA)-plasmin system in human umbilical vein endothelial cells (HUVECs) model.

METHODSTube formation assay was performed in vitro to evaluate the effects of BDNF on angiogenesis. The HUVECs were treated with various concentrations of BDNF (25 - 400 ng/ml) for different (6 - 48 hours), reverse transcriptase-polymerase chain reaction (RT-PCR) was used to assay MMP-2, MMP-9, TIMP-1, and TIMP-2 mRNA in HUVECs, and the conditioned medium was analyzed for MMP and uPA activity by gelatin zymography and fibrin zymography, respectively. uPA, plasminogen activator inhibitor (PAI)-1, tissue inhibitors of metalloproteinase (TIMP)-1, and TIMP-2 were quantified by western blotting analysis.

RESULTSBDNF elicited robust and elongated angiogeneis in two-dimensional cultures of HUVECs in comparison with control. The stimulation of serum-starved HUVECs with BDNF caused obvious increase in MMP-2 and MMP-9 mRNA expression and induced the pro-MMP-2 and pro-MMP-9 activation without significant differences in proliferation. However, BDNF had no effect on TIMP-1 and TIMP-2 production. BDNF increased uPA and PAI-1 production in a dose-dependent manner. Maximal activation of uPA and PAI-1 expression in HUVECs was induced by 100 ng/ml BDNF, while effects of 200 ng/ml and 400 ng/ml BDNF were slightly reduced in comparison with with those of 100 ng/ml. Protease activity for uPA was also increased by BDNF in a dose-dependent manner. BDNF also stimulated uPA and PAI-1 production beyond that in control cultures in a time-dependent manner from 12 hours to 48 hours after BDNF treatment.

CONCLUSIONSBDNF stimulates MMP and uPA/PAI-1 proteolytic network in HUVECs, which may be important to the acquisition of proangiogenic potential.

Brain-Derived Neurotrophic Factor ; pharmacology ; Cells, Cultured ; Gene Expression Regulation ; drug effects ; Humans ; Matrix Metalloproteinase 2 ; genetics ; Matrix Metalloproteinase 9 ; genetics ; Neovascularization, Physiologic ; drug effects ; Plasminogen Activator Inhibitor 1 ; genetics ; RNA, Messenger ; analysis ; Urokinase-Type Plasminogen Activator ; genetics

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

OBJECTIVEArrested lung development and lung fibrosis are characteristic pathological changes in chronic lung disease (CLD). Therefore, the role of extracellular matrix (ECM) remodeling in lung fibrosis has been emphasized recently. Plasmin system is also an important factor to modulate ECM degradation and matrix metalloproteinase (MMP) system. In this study, the authors established an animal model of CLD induced by inhaling high concentration oxygen (hyperoxia) to investigate the changes and functions of urokinase-plasminogen activator (u-PA) and plasminogen activator inhibitor-1 (PAI-1) in CLD.

METHODSFull-term newborn rats were continuously exposed to oxygen (0.90 - 0.95 O(2)) or room air within 12 h after birth. On day 1, 3, 7, 14, 21 in hyperoxia groups and air controls, lung pathology in newborn rats were observed. The changes of u-PA and PAI-1 protein and mRNA expression were measured by Western blotting and RT-PCR.

RESULTS(1) The pathological findings of the lung tissue: on day 3, there was a few inflammatory cells exuded out, bleeding, edema, and interstitial cells increased in hyperoxia group. On day 7 and thereafter, the terminal air space size of the oxygen-exposed rat became large, there was inflammatory response and more interstitial cells, interstitium was thicker, and collagen deposited. (2) u-PA expression: On day 3, the u-PA protein expression increased in hyperoxia group compared with controls (115.52 +/- 7.10 vs. 96.51 +/- 6.33), P < 0.01. On day 7 to day 21, u-PA protein expression (97.66 +/- 7.98, 99.91 +/- 7.60, 103.23 +/- 6.24) was lower than in the control groups (112.43 +/- 6.01, 123.25 +/- 8.35, 103.23 +/- 6.24), P < 0.05, < 0.01 and < 0.01, respectively. u-PA mRNA increased on d 3 in hyperoxia group compared with controls (1.18 +/- 0.07 vs. 0.99 +/- 0.05), P < 0.01. On d 7 to 21, mRNA expression (1.01 +/- 0.06, 1.10 +/- 0.12, 1.27 +/- 0.06) was lower than that in the controls (1.15 +/- 0.08, 1.51 +/- 0.32, 1.60 +/- 0.24) too, P < 0.01. (3) PAI-1 expression: From d 7 to 21 of oxygen exposure, PAI-1 protein expression (147.83 +/- 12.27, 149.07 +/- 11.17, 161.42 +/- 13.08) increased compared with the controls (116.18 +/- 10.67, 113.73 +/- 15.58, 126.60 +/- 8.59), P < 0.01, < 0.05 and < 0.01, respectively. mRNA expression (1.49 +/- 0.28, 1.46 +/- 0.31, 1.51 +/- 0.33) increased compared with the control group (0.94 +/- 0.01, 0.94 +/- 0.03, 0.98 +/- 0.03), P < 0.05, < 0.01 and < 0.05, respectively.

CONCLUSIONSIn the early stage of hyperoxic exposure, the balance of u-PA/PAI-1 mRNA and protein increased, plasmin and degradation activity increased, which may increase the degradation of ECM in lung base membrane. During the middle and late stage, the expression of u-PA/PAI-1 mRNA and protein decreased, plasmin and degradation activity were lower, in parallel to thicker interstitium, suggesting that the imbalance of u-PA/PAI-1 may also play a role in lung fibrosis in CLD induced by hyperoxia.

Animals ; Female ; Hyperoxia ; complications ; Lung ; metabolism ; pathology ; Lung Diseases, Interstitial ; etiology ; metabolism ; pathology ; Male ; Plasminogen Activator Inhibitor 1 ; genetics ; metabolism ; RNA, Messenger ; genetics ; Rats ; Rats, Wistar ; Urokinase-Type Plasminogen Activator ; genetics ; metabolism

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

OBJECTIVETo study the duration of prourokinase gene expression in vein grafts and the role of the prourokinase gene in protecting vein grafts from neointimal hyperplasia.

METHODSFifty-four Wistar rats were used in this study. In each rat, the jugular vein was excised and distended for 30 minutes using a solution containing either Adv(5)-CMV (control group) or Adv(5)-CMV/Pro-UK (treatment group). Next, the jugular vein was reversed and interposed into the divided carotid artery of the same rat. On the 14th day after transfection, vein grafts of the control group were collected in order to perform a fibrinolysis test for prourokinase (Pro-UK) activity. On the 2nd, 7th, 14th, 28th, and 60th day, the vein grafts of the treatment group were likewise collected in order to detect prourokinase activity. On the 28th day, the vein grafts of both groups were explanted to evaluate the (3)H-TDR incorporation so that pathologic analysis could be performed.

RESULTSPro-UK activity could not be detected in the control group, while in the treatment group, the Pro-UK activity could be detected from the 2nd day onwards, peaking on the 7th day and declining from the 14th day, but yet persisting at a low level for a further month. The amount of (3)H-TDR incorporated in the control group was higher than that in the treatment group. Pathologic analysis demonstrated that vein grafts of both groups exhibited wall thickening, but that the degree of graft neointimal hyperplasia and reduction of the graft lumen was greater in the control group than that in the treatment group. The occlusion rate of grafts in the control group was 20%. All grafts in the treatment group were patent.

CONCLUSIONSPro-UK gene transfer before vein grafting in vitro results in a high level of gene expression in the vein graft from the 7th day to 14th day. And its gene expression in the vein graft could reduce neointimal hyperplasia in the vein graft.

Animals ; Gene Expression ; Gene Transfer Techniques ; Hyperplasia ; Jugular Veins ; transplantation ; Male ; Rats ; Rats, Wistar ; Recombinant Proteins ; genetics ; Tunica Intima ; pathology ; Urokinase-Type Plasminogen Activator ; genetics ; Veins ; pathology ; transplantation

Taking a suspension adapted recombinant CHO cell line, 11G-S expressing human Pro-urokinase (Pro-UK) as the object of study, the impacts of different feeding nutrients, the start time of feeding and cell inoculation density on the growth and Pro-UK production of 11G-S cells in serum-free fed-batch culture were evaluated in 100 mL shacking flasks. The results indicated that amino acids, serum-free supplements and inorganic salts played important role in cell growth, cell viability and protein expression. And the effects of cells fed-batch culture was much better with the initial cell inoculation density at 3 x 10(5)-4 x 10(5) cells/mL and the start time of feeding set at 72 h, a maximum viable cells density of 7.8 x 10(6) cells/mL with a peak Pro-UK activity at 8570 IU/mL was achieved during 12 d fed-batch culture. Further, the mu of the 11G-S cells at the middle phase of the fed-batch culture, and both the q(glu) and q(gln) of the 11G-S cells at the middle and later phases of the fed-batch culture was higher than that of the 11G-S cells at the same phase of the batch culture, respectively.
Animals ; CHO Cells ; Cell Culture Techniques ; methods ; Cricetinae ; Cricetulus ; Culture Media, Serum-Free ; Humans ; Recombinant Proteins ; biosynthesis ; genetics ; metabolism ; Urokinase-Type Plasminogen Activator ; biosynthesis ; genetics ; metabolism