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 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

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 observe the effect of rhubarb in treating secondary damage of central nerve system (CNS) in rats with acute hemorrhagic stroke (AHS) and to explore the possible mechanism.

METHODSThe rat's AHS model was established by autologous blood injection, the effect of rhubarb on the secondary damage of CNS, plasminogen (PLG) in brain and tissue type plasminogen activator (t-PA) were observed.

RESULTS(1) The nerve function deficit signs reappeared in about 70% model rats 4 - 6 days after modeling and reached the peak at day 6 - 8, scored as 1.63 +/- 0.72 on day 4 and as 2.32 +/- 1.12 on day 7; (2) Rhubarb could effectively improve the secondary nerve function damage, with the nerve deficit scores kept to 1.24 +/- 0.19 from day 4 on, and no sign of secondary CNS damage was shown. The nerve deficit score was 1.22 +/- 0.15 on day 7 in the rhubarb treated group, showing significant difference as compared with that in the model group (P<0.05); (3) The specific amplified products of t-PA mRNA on day 3 and that of PLG mRNA on day 7 in CNS of model group were significantly higher than those in the sham operated group and the rhubarb treated group.

CONCLUSIONRhubarb could effectively reduce the secondary CNS damage in rats with AHS, it might be related with the suppressive effect of rhubarb on tPA mRNA and PLG mRNA expression in CNS.

Animals ; Drugs, Chinese Herbal ; pharmacology ; Intracranial Hemorrhages ; blood ; pathology ; physiopathology ; Male ; Plasminogen ; biosynthesis ; genetics ; Plasminogen Activator Inhibitor 1 ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Rats ; Rats, Sprague-Dawley ; Rheum ; chemistry ; Stroke ; blood ; pathology ; physiopathology

The plasminogen and plasmin system, which is mainly regulated by urokinase-type plasminogen activator (uPA), its receptor (uPAR) and its inhibitor (PAI-1), is generally believed to play a role in cancer invasion and metastasis. This study was conducted to investigate the role of uPA, uPAR and PAI-1 in the invasion and metastasis of gastric adenocarcinoma. The expression of mRNAs for uPA and PAI-1 was determined by Northern blot analysis in nine primary gastric cancer tissues, nine paired metastatic lymph nodes and normal gastric mucosa. The mRNA of uPA was not or faintly detected in normal mucosa, while the expression was increased in both primary gastric cancer tissues and metastatic lymph nodes to a similar degree. The mRNA expression for PAI-1 in the gastric cancer tissues was not different from that in the paired metastatic lymph nodes and normal mucosae. uPAR was determined by immunohistochemical staining, demonstrating that five (56%) and six (67%) out of nine primary gastric cancer tissues and nine paired metastatic lymph nodes were positive, respectively and the intensity was stronger in metastatic lymph nodes. The results support the concept that most gastric cancer cells may have an innately moderate level of uPA and uPAR, and that increase of uPAR expression can be considered to be closely associated with cancer invasion and metastasis.
Adenocarcinoma/*metabolism/pathology ; Gastric Mucosa/metabolism ; Gene Expression ; Human ; Immunoenzyme Techniques ; Lymph Nodes/metabolism/pathology ; Neoplasm Metastasis ; Plasminogen Activator Inhibitor 1/*biosynthesis/genetics ; Plasminogen Activators/*biosynthesis/genetics ; RNA, Messenger/biosynthesis ; Receptors, Cell Surface/*biosynthesis/genetics ; Stomach Neoplasms/*metabolism/pathology ; Support, Non-U.S. Gov't ; Urinary Plasminogen Activator/*biosynthesis/genetics

OBJECTIVE: To determine the mechanism of peritoneal fibrosis of peritoneal mesothelial cells by high glucose. METHODS: The third passage human peritoneal mesothelial cells (HPMCs) from primary culture were divided into a control group (F(12)) and high glucose groups (F(12)+4% glucose) in different times (24, 48 h). The cell proliferation was assayed by the method of MTT (methylthiazoletetrazolium). The cell damage was measured by LDH (lactate dehydrogenase). The protein expression of fibronectin (FN), transforming growth factor-beta1(TGF-beta(1)) and connective tissue growth factor (CTGF) were detected by ELISA. The mRNA expression of FN, TGF-beta(1) and PAI-1 were detected by RT-PCR. RESULTS: High glucose suppressed the cell proliferation. The result of MTT showed that compared with the control group, the value of OD of high glucose groups at 24 or 48 h decreased significantly (P<0.01 or 0.01); The cell damage was enhanced in high glucose groups, at 24 or 48 h compared with the control group at the same time (all P<0.01). The protein expressions of TGF-beta(1), CTGF and FN in supernate fluid of cell culture were significantly enhanced when high glucose stimulated the HPMCs in the high glucose groups at 24 or 48 h compared with the control group at the same time (P<0.05 or 0.001). The expressions of FN, TGF-beta(1) and PAI-1 mRNA were upregulated in 24 h high glucose group compared with that of 24 h control group. CONCLUSION High glucose can suppress the HPMC proliferation and damage HPMCs. Increase of TGF-beta(1), CTGF, FN and PAI-1 of HPMCs stimulated by high glucose can promote the synthesis and decreased degradation of extracellular matrix, which might be related with the mechanism of peritoneal fibrosis of peritoneal mesothelial cells by high glucose.
Cell Proliferation ; drug effects ; Cells, Cultured ; Epithelial Cells ; metabolism ; pathology ; Fibronectins ; biosynthesis ; genetics ; Glucose ; pharmacology ; Humans ; Peritoneal Dialysis ; Peritoneum ; metabolism ; pathology ; Plasminogen Activator Inhibitor 1 ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Transforming Growth Factor beta ; biosynthesis ; genetics

OBJECTIVETo explore the mechanism of anisodamine in treating infectious shock through studying effect of anisodamine on endotoxin lipopolysaccharide (LPS) induced expression of tissue factor (TF) and plasminogen activator inhibitor type 1 (PAI-1) in vascular endothelial cells (EC).

METHODSHuman umbilical vein endothelial cells (HUVEC) were cultured by trypsin digestion method. PAI-1 was measured in the conditioned medium of HUVEC by a specific enzyme-linked immunosorbent assay (ELISA), whereas TF activity was measured in the lysates of these cells by using a single step clotting assay. Specific mRNA expressions were determined by Northern blotting. In order to evaluate a possible contribution of the nuclear factor-kappa B (NF-kappa B) pathway on the transductive effects observed, electrophoretic mobility shift assays (EMSA) were performed using nuclear extracts from HUVEC and NF-kappa B binding oligonucleotides.

RESULTSLPS could significantly strengthen the expression of HUVEC PAI-1 protein and TF activity and its mRNA, this effect of LPS could be markedly weakened after adding Anisodamine dose-dependently. Anisodamine could also completely block the LPS induced NF-kappa B DNA binding activity in nuclear extracts from HUVEC.

CONCLUSIONThe possible mechanism of anisodamine in treating infectious shock may be through antagonizing LPS induced HUVEC TF and PAI-1 expression, and the antagonism might be, at least partially, transduced by path of NF-kappa B.

Cells, Cultured ; Culture Media, Conditioned ; Drugs, Chinese Herbal ; pharmacology ; Endothelial Cells ; cytology ; metabolism ; Humans ; NF-kappa B ; metabolism ; Plasminogen Activator Inhibitor 1 ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Solanaceous Alkaloids ; pharmacology ; Thromboplastin ; biosynthesis ; genetics ; Umbilical Veins ; cytology

OBJECTIVETo study the effect of nuclear transcription factor AP-1 on tumor necrosis factor alpha (TNF-alpha) or minimal modified low density lipoprotein (mmLDL)-induced expression of plasminogen activator inhibitor-1 (PAI-1) in human vascular endothelial cells.

METHODSUsing gene recombination techniques, four luciferase reporter gene plasmids containing different length of human PAI-1 gene promoter were constructed. Through the transient transfection analysis, the roles of AP-1 element (from -823 bp to -553 bp) in PAI-1 promoter have been determined. In order to further verify the role of AP-1 element, the three site-directed mutants were recovered using PCR and sequencing assay.

RESULTSThe induction by TNF-alpha or mmLDL were decreased markedly when the three AP-1 elements in PAI-1 promoter had been mutated respectively.

CONCLUSIONSThese results indicate that the AP-1 element in PAI-1 promoter may have important role in PAI-1 gene transcriptions in endothelial cells induced by TNF-alpha or mmLDL.

Base Sequence ; Cells, Cultured ; Endothelium, Vascular ; cytology ; metabolism ; Humans ; Molecular Sequence Data ; Plasminogen Activator Inhibitor 1 ; biosynthesis ; genetics ; Promoter Regions, Genetic ; Transcription Factor AP-1 ; genetics ; metabolism ; Tumor Necrosis Factor-alpha ; genetics ; metabolism ; Umbilical Veins ; cytology

OBJECTIVETo investigate the effect of testosterone with varied concentrations on the tPA and PAI-1 mRNA levels of Human umbilical vein endothelial cells (HUVEC).

METHODSHUVEC within 2 - 3 passages were cultured with testosterone (3, 30, 3 x 10(3), 3 x 10(4) nmol/L) , and the control confluent cells were cultured in the same medium without steroid for 48 hours. RT-PCR was carried out to detect tPA and PAI-1 mRNA levels.

RESULTStPA mRNA level increased, while PAI-1 mRNA levels decreased significantly, at the testosterone concentrations ranging from 3 to 3 x 10(3) nmol/L (P < 0.05). Both tPA and PAI-1 mRNA level decreased obviously of 3 x 10(4) nmol/L group.

CONCLUSIONThe results indicated that testosterone could stimulate tPA gene expression, while decreased PAI-1 mRNA level of HUVEC, which suggested that testosterone might have beneficial effects on preventing male's thrombotic diseases.

Cells, Cultured ; Dose-Response Relationship, Drug ; Endothelial Cells ; drug effects ; metabolism ; Endothelium, Vascular ; cytology ; metabolism ; Humans ; Male ; Plasminogen Activator Inhibitor 1 ; biosynthesis ; genetics ; RNA, Messenger ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Testosterone ; pharmacology ; Tissue Plasminogen Activator ; biosynthesis ; genetics ; Umbilical Veins ; cytology

OBJECTIVE[corrected] To investigate the effect of peroxisome proliferator-activated receptors (PPARs) activators on plasminogen activator inhibitor type-1 (PAI-1) expression in human umbilical vein endothelial cells and the possible mechanism.

METHODSHuman umbilical vein endothelial cells (HUVECs) were obtained from normal fetus, and cultured conventionally. Then the HUVECs were exposed to test agents (linolenic acid, linoleic acid, oleic acid, stearic acid and prostaglandin J2 respectively) in varying concentrations with fresh media. RT-PCR and ELISA were applied to determine the expression of PPARs and PAI-1 in HUVECs.

RESULTSPPAR alpha, PPAR beta and PPAR gamma mRNA were detected by using RT-PCR in HUVECs. Treatment of HUVECs with PPARalpha and PPAR gamma activators--linolenic acid, linoleic acid, oleic acid and prostaglandin J2 respectively, but not with stearic acid could augment PAI-1 mRNA expression and protein secretion in a concentration-dependent manner. However, the mRNA expressions of 3 subclasses of PPAR with their activators in HUVECs were not changed compared with controls.

CONCLUSIONHUVECs express PPARs. PPARs activators may increase PAI-1 expression in ECs, but the underlying mechanism remains unclear. Although PPARs expression was not enhanced after stimulated by their activators in ECs, the role of functionally active PPARs in regulating PAI-1 expression in ECs needs to be further investigated by using transient gene transfection assay.

Cells, Cultured ; Endothelium, Vascular ; cytology ; metabolism ; Fatty Acids ; pharmacology ; Fetus ; Humans ; Linoleic Acid ; pharmacology ; Plasminogen Activator Inhibitor 1 ; biosynthesis ; genetics ; Prostaglandin D2 ; analogs & derivatives ; pharmacology ; RNA, Messenger ; genetics ; Receptors, Cytoplasmic and Nuclear ; biosynthesis ; genetics ; Transcription Factors ; biosynthesis ; genetics ; Umbilical Veins ; cytology ; metabolism ; alpha-Linolenic Acid ; pharmacology