Gene transfer of basic fibroblast growth factor (bFGF) has been shown to induce significant endothelial migration and angiogenesis in ischemic disease models. Here, we investigate what factors are secreted from skeletal muscle cells (SkMCs) transfected with bFGF gene and whether they participate in endothelial cell migration. We constructed replication-defective adenovirus vectors containing the human bFGF gene (Ad/bFGF) or a control LacZ gene (Ad/LacZ) and obtained conditioned media, bFGF-CM and LacZ-CM, from SkMCs infected by Ad/bFGF or Ad/LacZ, respectively. Cell migration significantly increased in HUVECs incubated with bFGF-CM compared to cells incubated with LacZ-CM. Interestingly, HUVEC migration in response to bFGF-CM was only partially blocked by the addition of bFGF-neutralizing antibody, suggesting that bFGF-CM contains other factors that stimulate endothelial cell migration. Several proteins, matrix metalloproteinase-1 (MMP-1), plasminogen activator inhibitor-1 (PAI-1), and cathepsin L, increased in bFGF-CM compared to LacZ-CM; based on 1-dimensional gel electrophoresis and mass spectrometry. Their increased mRNA and protein levels were confirmed by RT-PCR and immunoblot analysis. The recombinant human bFGF protein induced MMP-1, PAI-1, and cathepsin L expression in SkMCs. Endothelial cell migration was reduced in groups treated with bFGF-CM containing neutralizing antibodies against MMP-1 or PAI-1. In particular, HUVECs treated with bFGF-CM containing cell-impermeable cathepsin L inhibitor showed the most significant decrease in cell migration. Cathepsin L protein directly promotes endothelial cell migration through the JNK pathway. These results indicate that cathepsin L released from SkMCs transfected with the bFGF gene can promote endothelial cell migration.
Antibodies, Neutralizing/immunology ; Cathepsin L/genetics/*metabolism ; *Cell Movement ; Cells, Cultured ; Comet Assay ; Dependovirus/genetics ; Endothelial Cells/cytology/*metabolism ; Fibroblast Growth Factor 2/genetics/immunology/*metabolism ; Gene Transfer Techniques ; Humans ; Immunoblotting ; JNK Mitogen-Activated Protein Kinases ; Lac Operon/genetics ; Mass Spectrometry ; Matrix Metalloproteinase 1/biosynthesis/genetics ; Muscle, Skeletal/*metabolism ; Neovascularization, Physiologic ; Plasminogen Activator Inhibitor 1/biosynthesis/genetics ; RNA, Messenger/biosynthesis ; Reverse Transcriptase Polymerase Chain Reaction

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 Salvia miltiorrhiza on glomerulosclerosis induced by Ang II.

METHODRat mesangial cells were exposed to 100 nmol L(-1) Ang II. Meanwhile, we added S. miltiorrhiza injection of different concentrations to Mcs. PAI-1 mRNA and protein, TGF-beta1 in serum free MEM medium, and the level of cellular reactive oxygen species (ROS) were measured.

RESULTS. miltiorrhiza notably attenuated Ang II induced expression of PAI-1 in a concentration-dependent manner. Meanwhile, S. miltiorrhiza suppressed the production of TGF-beta1 and cellular ROS in mesangial cells.

CONCLUSIONS. miltiorrhiza can alleviate glomerular sclerosis. The renoprotective effects of S. miltiorrhiza may be due to its ability to decrease Ang II -induced PAI-1 and TGF-beta1 secretion and cellular ROS level.

Angiotensin II ; pharmacology ; Animals ; Blotting, Western ; Cells, Cultured ; Dose-Response Relationship, Drug ; Drugs, Chinese Herbal ; administration & dosage ; isolation & purification ; pharmacology ; Gene Expression ; drug effects ; Injections ; Mesangial Cells ; drug effects ; metabolism ; Plants, Medicinal ; chemistry ; Plasminogen Activator Inhibitor 1 ; biosynthesis ; genetics ; RNA, Messenger ; genetics ; metabolism ; Rats ; Reactive Oxygen Species ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Salvia miltiorrhiza ; chemistry ; Transforming Growth Factor beta1 ; blood ; secretion

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

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 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

OBJECTIVETo evaluate whether the medicinal serum of Yi-shen Ruan-jian san can antagonize the fibrogenic effect of human proximal tubular epithelial cell line (HKC) activated by aristolochic acid (AA) in vitro.

METHODThe HKC was incubated in the media containing 40 mg x L(-1) aristolochic acid sodium salt (AA-Na) with or without 10% concentration of Yi-shen Ruan-jian san medicinal serum. Then the cell proliferation and cytotoxicity of HKC were determined by MTF and lactate dehydrogenase (LDH) release assay respectively, the antigen expression of cytokeratin and alpha-smooth muscle actin on HKC was detected by immunocytochemistry, the mRNA expression of transforming growth factor-beta1 (TGF-beta1), connective tissue growth factor (CTGF), plasminogen activator inhibitor-1 (PAI-1), tissue inhibitor of metalloproteinase-1 (TIMP-1) and type I Collagen (Col I) of HKC was measured by RT-PCR, and their protein expression was measured by ELISA or Western blot.

RESULTNo cytotoxic effect was found in HKC after stimulation of AA-Na with or without the medicinal serum of Yi-shen Ruan-jian san (P > 0.05). No epithelial-myofibroblast transdifferentiation was found in HKC after AA-Na stimulation. The mRNA and protein expression of TGF-beta1, CTGF, PAI-1 and TIMP-1 of HKC was significantly upregulated by AA-Na (P < 0.05). The above-mentioned enhanced mRNA and protein expression, except for PAI-1, was significantly downregulated by the medicinal serum of Yi-shen Ruan-jian san, compared with the control (normal rat serum in the same concentration) (P < 0.05).

CONCLUSIONThe fibrogenic effects of HKC activated by AA are antagonized by Yi-shen Ruan-jian san, through downregulating the expression of promoting excellular matrix (ECM) synthesis factors (TGF-beta1, CTGF) and inhibiting ECM degradation factor (TIMP-1).

Animals ; Aristolochic Acids ; antagonists & inhibitors ; toxicity ; Cell Line ; Cell Proliferation ; drug effects ; Connective Tissue Growth Factor ; Drug Combinations ; Drugs, Chinese Herbal ; pharmacokinetics ; pharmacology ; toxicity ; Epithelial Cells ; drug effects ; metabolism ; Immediate-Early Proteins ; biosynthesis ; genetics ; Intercellular Signaling Peptides and Proteins ; biosynthesis ; genetics ; Kidney Tubules, Proximal ; cytology ; L-Lactate Dehydrogenase ; metabolism ; Male ; Materia Medica ; pharmacology ; Plants, Medicinal ; chemistry ; Plasminogen Activator Inhibitor 1 ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Rats ; Rats, Sprague-Dawley ; Serum ; Tissue Inhibitor of Metalloproteinase-1 ; biosynthesis ; genetics ; Transforming Growth Factor beta ; biosynthesis ; genetics ; Transforming Growth Factor beta1

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

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

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