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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2006;26:2819.)
© 2006 American Heart Association, Inc.

Thrombosis

Tissue Factor Pathway Inhibitor-2 Is Upregulated by Vascular Endothelial Growth Factor and Suppresses Growth Factor-Induced Proliferation of Endothelial Cells

Zhenhua Xu; Debasish Maiti; Walter Kisiel; Elia J. Duh

From the Department of Ophthalmology (Z.X., D.M., E.J.D.), The Wilmer Ophthalmological Institute, Johns Hopkins University School of Medicine, Baltimore, Md; and the Department of Pathology (W.K.), University of New Mexico Health Sciences Center, Albuquerque, NM.

Correspondence to Elia J. Duh, MD, Department of Ophthalmology, The Johns Hopkins University School of Medicine, 1550 Orleans Street, Baltimore, MD 21231. E-mail eduh{at}jhmi.edu

Objective— The purpose of this study is to investigate the expression and regulation of type-2 tissue factor pathway inhibitor (TFPI-2) in endothelial cells, as well as the regulation of human endothelial cell (EC) function by TFPI-2.

Methods and Results— Real-time polymerase chain reaction (PCR) and Western blot analysis revealed that vascular endothelial growth factor (VEGF) induced both time- and dose-dependent increase in TFPI-2 mRNA and protein expression in endothelial cells. TFPI-2 mRNA expression was also significantly upregulated by IL-1ß, and modestly increased by both tumor necrosis factor (TNF)- and fibroblast growth factor (FGF)-2, but not placental growth factor (PlGF). VEGF upregulation of TFPI-2 was dramatically reduced by inhibition of the MEK pathway. Administration of TFPI-2 protein suppressed both VEGF and FGF-2 stimulation of EC proliferation in a dose-dependent manner. A recombinant preparation of the first Kunitz-type domain of TFPI-2 (KD1) did not suppress growth factor stimulation of EC proliferation, suggesting a mechanism distinct from the proteinase inhibitory activity of TFPI-2. Exogenously added TFPI-2 protein suppressed VEGF-induced EC migration in 2 different assays. Recombinant wt-KD1 or the R24K mutant of KD1, but not the R24Q mutant, dramatically suppressed VEGF-induced EC migration. TFPI-2 protein, but not recombinant KD1, blocked VEGF-induced activation of both Akt and ERK1/2 in ECs. At higher doses, TFPI-2 protein blocked VEGFR2 activation.

Conclusion— Our data suggest that VEGF-upregulation of TFPI-2 expression in endothelial cells may represent a mechanism for negative feedback regulation and modulation of its pro-angiogenic action on endothelial cells. TFPI-2, or derivatives of TFPI-2, may be novel therapeutics for treatment of angiogenic disease processes.

This study investigated the expression and regulation of type-2 tissue factor pathway inhibitor (TFPI-2) in endothelial cells, as well as the regulation of human endothelial cell (EC) function by TFPI-2. Our data suggest that VEGF upregulation of TFPI-2 expression in endothelial cells may represent a mechanism for negative feedback regulation and modulation of its pro-angiogenic action on endothelial cells. TFPI-2 may be a novel therapeutic for treatment of angiogenic disease processes.

Key Words: cell signaling • endothelial cells • migration • proliferation • tissue factor pathway inhibitor-2 • vascular endothelial growth factor

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