- Original basic research
- Open Access
Induction of Tissue Factor Expression in Endothelial Cells by Basic Fibroblast Growth Factor and its Modulation by Fenofibric acid
© Kaneko et al; licensee BioMed Central Ltd. 2003
- Received: 08 August 2003
- Accepted: 11 October 2003
- Published: 11 October 2003
Tissue factor (TF), expressed in endothelial cells (ECs) and enriched in human atherosclerotic lesions, acts as a critical initiator of blood coagulation in acute coronary syndrome. Basic fibroblast growth factor (bFGF) induces the proliferation and migration of ECs and plays a role in angiogenesis and restoration of endothelial integrity. As TF is implicated in angiogenesis, we studied the effect of bFGF on TF gene and protein expression. Methods: Human umbilical vein ECs (HUVECs) were exposed to bFGF. TF mRNA was assessed by Northern blot and TF protein was assessed by Western blot. TF promoter activity was assessed by transient transfection assay and transcription factor was identified by electro mobility shift assay.
bFGF increased TF mRNA and protein expression in HUVECs. Increased TF mRNA was attenuated by inhibition of extracellular signal-regulated kinase kinase in human ECV304 cells. Transient transfection assays of the human TF promoter-luciferase construct (-786/+121 bp) demonstrated that bFGF induced transcription was dependent on the elements within the -197 to -176 bp relative to the transcription start site of the human TF gene. This region contains NF-κB like binding site. Electro mobility shift assay showed that bFGF increased nuclear translocation or DNA binding of NF-κB transcription factor to TF promoter. Nucleotide substitution to disrupt NF-κB like site reduced bFGF stimulated promoter activity. Fenofibric acid, an agonist ligand for the peroxisome proliferator activated receptor-α, reduced basal and bFGF stimulated TF expression.
These results indicate that bFGF may increase TF production in ECs through activation of transcription at NF-κB binding site, and control coagulation in vessel walls. Fibrate can inhibit TF expression and therefore reduce the thrombogenecity of human atherosclerotic lesions.
- tissue factor
Tissue factor (TF) is an integral membrane protein, which binds to coagulation factor VII/VIIa and initiates the coagulation cascade . TF expression can be inducible with inflammatory cytokines, lipopolysaccharide (LPS) and oxidized low-density lipoprotein in fibroblast, vascular smooth muscle cells and monocytes in human atherosclerotic lesions . TF expressed on the surface of vascular wall acts as the major procoagulant for thrombus formation .
Basic fibroblast growth factor (bFGF) elaborated by vascular cells can induce proliferation, morphological changes and migration of smooth muscle cells and endothelial cells (ECs) and modulate angiogenesis . bFGF promotes re-endothelialization with functional endothelium after balloon injury or induced atherosclerosis and modulates TF expression in monocytes and smooth muscle cells in vivo . In ECs TF expression is regulated through a promoter region, which encompasses potential binding sites for activator protein-1 (AP-1), nuclear factor (NF)-κB and SP-1 transcription factors . LPS-induced TF expression in cells of the monocyte lineage requires functional interaction between these transcription factors . However, the influence, if any, of bFGF on synthesis of TF in ECs has not been elucidated. The potential influence may well contribute to physiologic or pathophysiologic consequences of elaboration of bFGF.
The mechanisms inhibiting TF expression in ECs remain largely unexplored. Fenofibric acid is a synthetic ligand for the peroxisome proliferator activated receptor (PPAR)-α . PPARα, as well as the other members of the PPAR family, are ligand-activated transcription factors belonging to the nuclear hormone receptor superfamily. Certain fatty acids and fibric acid derivative, which activates PPARα, reduces TF expression in human monocyte cells and macrophages in vitro . However, to the best of our knowledge, there has been no detailed documentation on the effect of PPARα on TF expression in ECs under both clinical and pathological settings. Therefore, in the present study we characterized the influence of bFGF and fibric acid on elaboration of TF in vitro by cultured ECs, and sought to delineate potential mechanisms that may underlie increased TF expression.
Human umbilical vein ECs (HUVECs) were obtained from Clonetics (Walkersville, MD). Human ECV304 cell line (ECV cells) was obtained from American Type Culture Collection (Manassas, VA). Penicillin-streptomycin solution, medium 199, Dulbecco's modified Eagle's media (DMEM) and trypsin were purchased from Sigma (St. Louis, MO). Calf serum was obtained from Hyclone (Logan, UT). PD98059 was from Research Biochemical International and GF109203X and genistein were from Sigma. All other chemicals were of the highest available commercial grade.
Cell culture procedures
HUVECs and ECV cells were cultured and stimulated with recombinant human bFGF (Genzyme, Minneapolis, MN) as previously described . HUVECs were incubated in DMEM without serum and exposed to bFGF for 24 hr. Cell lysates were prepared as previously described  and stored at -80°C. ECV cells were cultured in medium 199 containing 10% calf serum. In some experiments fenofibric acid (Kaken, Tokyo, Japan) was prepared as previously described , and ECV cells were preincubated with fenofibric acid for 24 hr before exposure to bFGF. Cell viability was determined by trypan blue exclusion and MTT assay (Sigma).
Assays for TF antigen by Western blotting
TF antigen was assayed by Western blotting as previously described . In brief, equivalent amounts of cell lysates were electrophoresed and transferred to polyvinylidene difluoride membranes. Membranes were incubated with 0.2 μg/ml mouse anti-human TF IgG (American Diagnostica, Greenwich, NY). Membranes were washed and incubated with alkaline-phosphatase conjugated guinea pig anti-mouse IgG (Sigma) diluted 1:5000. Membranes were incubated with chemiluminescent enhancer system (Immun-Star, Bio-Rad, Hercules, California) and exposed to XAR film (Kodak, Rochester, NY), which was then developed. Bands were quantified with the use of a densitometer and ImageQuant software (Molecular Dynamics, Sunnydale, CA).
Isolation of total RNA and Northern blotting
Total RNA was isolated by the acid guanidium thiocyanate-phenol-chloroform method. Northern blot analysis was performed as described previously . Gel-purified polymerase chain reaction (PCR) fragments for TF (-786 to +121 bp) and β-actin, radiolabeled with α-32P-dCTP by random priming, were used as probes. Autoradiography was performed and band intensities were determined by densitometric analysis.
Promoter-luciferase vector and expression plasmid
The human TF promoter 5' flanking region from -786 to +121 (907 bp) was amplified by PCR from human genomic DNA isolated from umbilical vein with the use of forward PCR primer 5'-AGAGGCAAACTGCCAGATGT-3' (-786 to -766) and the reverse primer 5'-TGTCTACCAGTTGGCGGCCG-3' (+101 to +121), respectively . The PCR product was gel-purified and subcloned into the multiple cloning sites of the pT7Blue T-Vector (Novagen, Madison, WI). The T-vector was digested with EcoR I and Hind III and subcloned into the multiple cloning sites of the promoterless Renilla luciferase reporter gene vector pRL-null (Promega, Madison, WI). Basal expression of luciferase activity of the TF promoter vector was detected by the TF Full (-786/+121) luciferase vector, which includes two AP-1, three SP-1 (Egr-1), and two NF-κB binding sites. The 5' deletion mutants were generated by PCR with the use of 5' primers complementary to the TF gene sequence. Deletion mutants of TF promoter vectors were constructed as follows: pTF 1F (-513/+121) luciferase lacks distal AP-1 like binding site, but retains two NF-κB like binding sites, proximal AP-1 like binding site and distal SP-1/Egr-1 like binding site. pTF 2F (-318/+121) luciferase lacks distal NF-κB like binding site, but retains proximal AP-1 like binding site, proximal NF-κB like binding site and distal SP-1/Egr-1 like binding site. pTF 3F (-197/+121) luciferase lacks both AP-1 like binding sites and distal NF-κB like binding site, but retains proximal NF-κB like binding site and distal SP-1/Egr-1 like binding site. pTF 4F (-176/+121) luciferase lacks proximal NF-κB like binding site, but retains distal SP-1/Egr-1 like binding site. pTF 5F (-129/+121) luciferase lacks distal SP-1/Egr-1 like binding site. Point mutation of TF promoter vectors was also constructed . Basal expression of luciferase activity of TF promoter vector was detected by the TF Full luciferase vector, which has normal proximal NF-κB like site (5'-CGGAGTTTCC-3'). One mutation vector has two point mutations in NF-κB like site as underlined (5'-CGGAGTTTGG-3') and another mutation vector has two point mutations in NF-κB like site (5'-CGGAGTTTGG-3') and one point mutation in SP-1/Egr-1 like site (5'-AGGCA GGGCAGGGGTG-3') as underlined.
DNA transfection and the luciferase assay
DNA transfection and the luciferase assay were performed as previously described . ECV cells of approximately 80% confluency were cotransfected with each TF promoter Renilla luciferase fusion DNA reporter construct (20 μg of pRL vector) and a Firefly luciferase pGL vector to control for transfection efficiency (20 μg) introduced by electroporation (240 V, 960 μFD). These cells were cultured in DMEM with 10% calf serum for 24 hours, stimulated with bFGF (10 ng/ml) in DMEM containing 10% calf serum for 24 hours and harvested. Cell lysate luciferase activity was measured with Dual-Luciferase Reporter Assay System (Promega). Normalized luciferase activity was calculated as the ratio of luciferase activity to control vector activity. Results for each reporter construct were expressed as fold induction compared with results in transfected, unstimulated cells.
Electrophoretic mobility shift assay
For electrophoretic mobility shift assay (EMSA) ECV cells were preincubated for 24 hours with fenofibrate 100 μmol/L and then stimulated for 2 hours with bFGF 100 ng/ml before nuclear extracts were prepared. The NF-κB oligonucleotide (ACGCGTCGGAGTTTCC TTGAACGGAGTTTCC GATC) spanning the 2 tandem NF-κB like sites (as underlined above) in the human TF promoter was end-labeled with [α-32P]dCTP (3000 Ci/mmol) by Klenow fragment of DNA polymerase I (Amersham Life Science, Buckinghamshire, England) and purified with NucTrap column (Stratagene, La Jolla, CA). Nuclear extracts (0.3 μg) were incubated with the labeled NF-κB like oligonucleotide under standard conditions. In the indicated experiments nuclear extracts were incubated with rabbit polyclonal anti-NFκB p50, rabbit polyclonal anti-NFκB p65 or mouse monoclonal anti-c-Rel IgG (Santa Cruz Biotechnology, Santa Cruz, CA) or nonspecific IgG before the addition of radiolabeled NF-κB probes. DNA-protein complexes were electrophoretically separated. Specificity was determined by addition of an excess of unlabeled NF-κB oligonucleotide to the nuclear extracts before formation of DNA-protein complexes.
Data are means ± SD. Differences were assessed by analysis of variance with Bonferroni's least significant post hoc tests for comparisons within multiple groups. Significance was defined as P < 0.05.
Effects of bFGF on TF expression in ECs
Effects of PD 98059 and GF109203X on TF mRNA
Determination of the DNA regions critical for basal and bFGF inducible TF transcriptional activity
Electrophoretic mobility shift assay
Effects of fenofibric acid
In this study bFGF was shown to influence production of TF in ECs. Increased TF protein correlated with increased mRNA and activity of the promoter. Fenofibric acid, an agonist ligand for PPARα, reduced basal and bFGF stimulated TF expression.
The binding of bFGF to its specific receptor triggers a cascade of events leading to signal transductions including ERK, mitogen-activated protein kinase, Ras and PKC pathways [12, 13]. The results in this study suggest that the induction mechanism of TF expression by bFGF may involve ERK kinase. These results are consistent with the recent study, which demonstrated that bFGF induced I-κB degradation and NF-κB activation through ERK1/2 pathway in smooth muscle cells . In addition, platelet-derived growth factor induces TF expression in smooth muscle cells by an ERK pathway-dependent mechanism and, in part, by Ras and PKC pathway-dependent mechanisms  and ERK activity is required for NF-κB activation by IL-1β . Our study demonstrated that bFGF could act in the same manner on TF expression in human ECs.
Deletion analysis of TF promoter revealed that a region between -197 bp and -176 bp contains the elements mediating the bFGF response. A search for the putative transcription factor binding sites within this region showed it to contain an NF-κB like site (CGGAGTTTCC) at -188 to -178 bp. Furthermore, basal promoter activity was regulated by this NF-κB like site and by SP-1/Egr-1 like site (GAGGCGGGGCAGGGGT) present at -169 to -154 bp. NF-κB, AP-1 and SP-1 elements in human TF promoter are involved in the activation of TF gene in ECs [6, 16]. Our results suggest that NF-κB element is important in TF gene activation after bFGF stimulation in ECs. In addition, SP-1/Egr-1 element is likely required for basal transcription from the TF promoter. The role of transcription repressor of the TF gene in ECs  needs further investigation.
PPARα negatively regulates vascular inflammatory gene response by negative cross-talk with NF-κB and AP-1 . PPARα is expressed in ECV cells . PPARα ligand, fenofibric acid, diminished both basal and bFGF inducible expression of TF, potentially reducing the thrombogenicity of atherosclerotic lesions. Deletion of the promoter region containing the NF-κB site inhibited the effect of bFGF and EMSA suggested that fenofibric acid did not inhibit nuclear translocation or DNA binding of the NF-κB like transcriptional factor to the TF promoter. Thus, inhibition of TF synthesis by fenofibric acid may occur transcriptionally as a result of PPARα repression of NF-κB signaling. Further studies are in progress to elucidate the mechanism of fenofibric acid effect using cells from arterial sources. These data indicate that activation of PPARα results in the downregulation of the TF gene and provide new insight into how PPARα-activating fibric acid derivatives might influence atherothrombosis in patients with vascular disease. The present study tested a novel role for PPARα in the control of atherosclerotic plaque thrombogenicity through its effects on TF expression in ECs and suggested that fibric acid may reduce thrombotic complications of atherosclerosis.
This work was supported by grants-in-aid for scientific research from the Ministry of Education, Science, Sport and Culture of Japan and from the Ministry of Welfare and Labor, and by a grant-in-aid from the Mitsui Life Social Welfare Foundation (Tokyo, Japan). The authors thank Ms. Kaori Abumiya, Mizuho Kasai and Akiko Aita for technical assistance.
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