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Angiopoietin-1 negatively regulates expression and activity of tissue factor in endothelial cells ¹

INJUNE KIM, JONG-LARK OH, YOUNG SHIN RYU, JUNE-NO SO^*, WILLIAM C. SESSA, KENNETH WALSH and GOU YOUNG KOH²

National Creative Research Initiatives Center for Endothelial Cells and Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang, 790–784;
^* Department of Biotechnology, Woosuk University, Chonju, 560–180, Republic of Korea;
Departments of Pharmacology, Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, Connecticut 06536-0812, USA; and
Division of Cardiovascular Research, St. Elizabeth’s Medical Center of Boston, Massachusetts 02135, USA

²Correspondence: National Creative Research Initiatives Center for Endothelial Cells, Division of Molecular and Life Sciences, Pohang University of Science and Technologym, San 31, Hyoja-Dong, Pohang, 790–784, Republic of Korea. E-mail: gykoh{at}postech.ac.kr

SPECIFIC AIM

Vascular endothelial growth factor (VEGF) has multiple cytokine functions in endothelial cells that are carried out through multiple intracellular signaling pathways whereas the other endothelial cell-specific ligand, angiopoietin-1 (Ang1), has varied affects on VEGF activity in endothelial cells. This study seeks to determine the role of Ang1 in VEGF- and tumor necrosis factor (TNF-) -induced expression of tissue factor in primary cultured endothelial cells and to reveal the pathway through which Ang1 fulfills this role.

PRINCIPAL FINDINGS

1. Ang1 inhibits the VEGF- and TNF--induced TF expression
We examined the effect of Ang1 on VEGF- and TNF--induced TF expression and activity in human umbilical vein endothelial cells (HUVECs). Ang1 (200 ng/ml) by itself did not produce any significant effect on TF mRNA level in HUVECs for up to 12 h (Fig. 1 , Fig. 2 ,;F1,2> anddata not shown). However, at 1 and 2 h, respectively, Ang1 (200 ng/ml) inhibited 65% and 62% of the VEGF-induced TF mRNA and 60% and 67% of the TNF--induced TF mRNA (Fig. 1) .

View larger version (50K): [in this window] [in a new window]   Figure 1. Ang1 suppresses VEGF- or TNF--induced TF mRNA in HUVECs. A, B) HUVECs were incubated with VEGF165 (20 ng/ml) or TNF- (10 ng/ml) for 1 or 2 h in the presence of control buffer (CB) or Ang1 (A1, 200 ng/ml). Total RNAs (10 µg) were subjected to RPA probed with an antisense TF RNA probe (221 bp). Equivalent loading was confirmed by simultaneously probing with an antisense cyclophilin RNA probe (105 bp). Spliced products of TF mRNA are detected between the TF and cyclophilin bands. Results were similar in 3 independent experiments. C, D) Densitometric analyses are presented as the relative ratio of TF mRNA to cyclophilin mRNA. The relative ratio measured for the CB is arbitrarily presented as 1. Results were similar in 3 independent experiments. Bars represent the mean ± SD from 3 experiments. *P < 0.05 vs. control buffer only. +P < 0.05 vs. VEGF165 or TNF- only at each time point.

View larger version (17K): [in this window] [in a new window]   Figure 2. Ang1 suppresses VEGF- or TNF--induced TF protein (A), TF activity (B), and procoagulant activity (C) in HUVECs. HUVECs were incubated with VEGF165 (20 ng/ml) or TNF- (10 ng/ml) for 8 h (for determination of TF protein and activity) or 12 h (for determination of procoagulant activity) in the presence of control buffer (CB) or Ang1 (A1, 200 ng/ml). TF activity or relative procoagulant activity of 1 is equivalent to 10-15 mol of TF (B, C). Results were similar in 5 independent experiments. Bars represent the mean ± SD from 5 experiments. *P < 0.05 vs. time 0 or control buffer only. +P < 0.05 vs. VEGF165 or TNF- only.

2. Ang1 inhibits the VEGF- and TNF--induced TF protein and activity
Ang1 inhibited 50%, 46%, and 33% of the VEGF-induced TF protein, TF cell surface activity, and procoagulant activity, respectively. Ang1 inhibited 40%, 40%, and 65% of the TNF--induced TF protein, TF cell surface activity, and procoagulant activity, respectively (Fig. 2) . A fivefold molar excess of rTie2-Fc, but not rTie1-Fc, completely blocked Ang1-induced suppression of VEGF-and TNF--induced TF protein, TF cell surface activity, and procoagulant activity (data not shown). These results indicate that Ang1 exerts its effects in endothelial cells through Tie2 receptor binding, but not through Tie1. Using flow cytometry, we also confirmed that Ang1 inhibited VEGF- and TNF--induced TF protein on the cell surface of HUVECs (data not shown).

CONCLUSIONS

TF is the primary cellular initiator of blood coagulation. TF is also involved in thrombosis and inflammation associated with sepsis, atherosclerosis, and cancer. TF can also participate in other processes, including metastasis, tumor-associated angiogenesis, and embryogenesis. Normally, TF is not expressed on the surface of endothelial cells. However a number of stimuli including TNF-, interleukin 1, bacterial lipopolysaccharide, thrombin, and VEGF induce TF expression. Thus, like TNF-, VEGF can act as a procoagulant cytokine by increasing TF expression in endothelial cells.

NF-B and AP-1 are major transcription factors for TNF--induced TF expression in endothelial cells. Although the intracellular signaling pathway responsible for VEGF-induced TF expression in endothelial cells has not yet been identified, reporter gene studies have implicated that transcription factors (including EGR-1, Sp1, and nuclear factor of activated T cells) are involved. However, our preliminary results demonstrated that pharmacological inhibition of PI 3'-kinase with wortmannin resulted in enhancement of VEGF- and TNF--induced expression of TF mRNA, protein, and activities in endothelial cells. These data are consistent with a recent report showing that inhibition of PI 3'-kinase with wortmannin enhanced TF expression induced by VEGF- and TNF-. Transcriptional activation by EGR-1, NF-B, and AP-1 may be essential positive components of TF expression whereas activation by PI 3'-kinase may be a negative suppressor for VEGF- and TNF--induced TF expression. In fact, VEGF and TNF- activate multiple intracellular signaling pathways in endothelial cells. Although VEGF and TNF- are known to activate the PI 3'-kinase pathway in endothelial cells, their activations are minimal compared to other signaling mediators, including the MEK/ERK and PLC/PKC pathways or NF-B activation. Therefore, the net effect of VEGF and TNF- is an increase in TF expression.

Because Ang1 is a strong activator of the intracellular PI 3'-kinase/Akt signaling system, we examined the effect of Ang1 on VEGF- and TNF--induced TF expression and activity in HUVECs. Our results demonstrate that Ang1 not only inhibits VEGF- and TNF--induced expression of TF, but also inhibits VEGF- and TNF--induced TF activity and procoagulant activity of primary cultured endothelial cells. Our biochemical study indicates that Ang1 induces Akt phosphorylation at Ser473; this induction depends on PI 3'-kinase and is much stronger and more persistent than VEGF-induced Akt phosphorylation. Our findings also indicate that Ang1 strongly induces Akt phosphorylation at Ser473 in the presence of VEGF and TNF-. Thus, Ang1-induced activation of PI 3'-kinase/Akt could be a crucial step in Ang1’s suppressive effect on VEGF- and TNF--induced expression and on the activity of TF in endothelial cells. To address the question of whether direct activation of PI 3'kinase/Akt inhibits TF expression, we used adenoviral gene transfer to deliver genes that express constitutively active and dominant-negative forms of PI 3'-kinase and Akt in primary cultured endothelial cells. Constitutive activation of PI 3'-kinase or Akt profoundly reduced TNF--induced expression of TF mRNA and protein, whereas dominant-negative inactivation of PI 3'-kinase or Akt profoundly enhanced TNF--induced expression of TF mRNA and protein. From these results, we conclude that Ang1 reduces VEGF- and TNF--induced TF expression through intracellular activation of the PI 3'-kinase/Akt pathway (Fig. 3) .

View larger version (17K): [in this window] [in a new window]   Figure 3. Possible pathways for Ang1 inhibition of VEGF- and TNF--induced TF expression and activity in endothelial cells. Transcriptional activation by EGR-1, Sp-1, NFAT, NF-B, or AP-1 is involved in VEGF- and TNF--induced TF expression and activity. Activation of intracellular PI 3'-kinase/Akt by Ang1 may suppress transcriptional activity or TF mRNA level.

We have recently shown how VEGF stimulates adhesion in HUVECs. VEGF stimulated the expression of ICAM-1, VCAM-1, and E-selectin mRNAs, and this induction occurred mainly through activation of PLC and NF-B. The induction was suppressed by activation of PI 3'-kinase. Our recent report indicated that Ang1-induced activation of PI 3'-kinase/Akt suppressed VEGF-induced expression of adhesion molecules. Therefore, contrary to previous observations in embryonic kidney 293 cells and fibroblasts, activation of PI 3'-kinase/Akt may suppress the transcriptional activation of NF-B in endothelial cells. Thus, the pathway through which Ang1-induced activation of PI 3'-kinase/Akt suppresses the transcriptional activation of NF-B in endothelial cells remains to be determined.

In summary, we found a novel effect of Ang1 on VEGF- and TNF--induced expression and activity of TF in endothelial cells, and shed light on the underlying mechanism by which Ang1 suppresses VEGF- and TNF--induced expression and activity of TF. Our results suggest that Ang1 suppresses VEGF- and TNF--induced TF expression through intracellular activation of the PI 3'-kinase/Akt pathway (Fig. 3) . Thus, Ang1 may have clinical application as a therapeutic inhibitor of VEGF- and TNF--induced coagulation, inflammation, and cancer progression.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0556fje; to cite this article, use FASEB J. (November 29, 2001) 10.1096/fj.01-0556fje

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