Adenovirus-Mediated Expression of Tissue Factor Pathway Inhibitor-2 Inhibits Endothelial Cell Migration and Angiogenesis

Abstract

Objective-Extracellular matrix (ECM) remodeling during angiogenesis is accomplished through plasmin-dependent pericellular proteolysis and through the action of matrix metalloproteinases (MMPs). Because tissue factor pathway inhibitor-2 (TFPI-2), a Kunitz-type protease inhibitor with prominent ECM localization, inhibits plasmin and MMPs activity, we investigated the role of TFPI-2 in endothelial cell (EC) migration and angiogenesis. Methods and Results-Real-time polymerase chain reaction and immunostaining showed that the expression of TFPI-2 mRNA and protein was upregulated in migrating ECs. The effect of TFPI-2 on angiogenesis was studied in mouse models of Matrigel and polyvinylalcohol sponge implants by overexpressing TFPI-2 through infection with a replication-deficient adenovirus (AdTFPI-2). Using (immuno)fluorescence and confocal microscopy we observed that TFPI-2 reduced neovascularization and promoted ECM deposition. Lateral cell migration and capillary tube formation in vitro also were impaired by TFPI-2, a process reversed by anti-TFPI-2 antibodies. Increased apoptosis occurred both in AdTFPI-2-treated ECs and in the mouse implants. Zymography and assays in the absence of plasminogen confirmed plasmin inhibition as a main mechanism through which TFPI-2 inhibits EC migration. Conclusions-Our data suggest that TFPI-2 may be an important regulator of aberrant angiogenesis associated with tumor growth/metastasis, cardiovascular diseases, chronic inflammation, or diabetes. Key Words: TFPI-2 Ⅲ angiogenesis Ⅲ endothelial cell Ⅲ adenovirus gene delivery Ⅲ mouse model P roteinase inhibitors are involved in blood coagulation, fibrinolysis, angiogenesis, wound healing, and tumor invasion. 1 Tissue factor pathway inhibitor-2 (TFPI-2) is a Kunitz-type serine proteinase inhibitor 2 synthesized by endothelial cells (ECs), smooth muscle cells (SMCs), and syncytiotrophoblasts, 3,4 which associates through ionic interactions 5 with the extracellular matrix (ECM), 6 and inhibits trypsin, plasmin, and plasma kallikrein among others. 7 The ECM is essential for the integrity of the cardiovascular system. ECs use the matrix as scaffolding during invasion, but they also degrade it to provide space for new capillaries. Serine proteases belonging to the plasminogen activator (PA)/plasmin system and (membrane-type)-matrix metalloproteinases [(MT)-MMPs] play major roles in all the steps of angiogenesis 8 : vessel sprouting, cell migration, tube formation, survival, generation of matrikines as angiogenesis inhibitors, and vessel stabilization/maturation and remodeling. 9,10 Matrix metalloproteinase (MMP)-2, MMP-9, and MT1-MMP switch on neovascularization through degradation of the endothelial and interstitial matrix, and activation of growth factors (reviewed in 10 ). Protease activities are controlled by specific activation mechanisms and inhibitors, of which tissue inhibitors of MMPs and serpins, like PA inhibitors (PAIs), represent major classes. MMPs are secreted as inactive zymogens that are activated by other proteinases, s...

Human tissue factor pathway inhibitor-2 induces caspase-mediated apoptosis in a human fibrosarcoma cell line

“…While others have demonstrated apoptosis induction in EA.hy926 endothelial cells, as well as glioblastoma, malignant meningioma and prostate tumor cell lines through overexpression of TFPI-2 [11,[14][15][16][17], it is unclear whether these cells undergo apoptosis following exogenous application of either TFPI-2 or R24K KD1. To this end, we incubated human umbilical vein endothelial cells, a human colon carcinoma cell line (Colo-205) and a human bladder carcinoma cell line (J-82) with either vehicle, 1 lM TFPI-2, 1 lM R24K KD1 or 1 lM R24Q KD1 for 48 h. Following incubation, the cells were processed for EB/AO staining and immunoblotting of the cell lysate to quantify the level of activated caspase-3, since the activation of caspase-3 serves as a sensitive marker of apoptosis [20].…”

“…In addition, overexpression of TFPI-2 in several highly invasive tumor cells has been shown to inhibit their growth, invasiveness, angiogenic and metastatic potential [6,[11][12][13][14]. Finally, recent studies have shown that adenovirus vector-mediated overexpression of TFPI-2 inhibits EA.hy926 endothelial cell migration and angiogenesis, and also increased apoptosis of these cells by over twofold [15].…”

Foxk1 promotes cell proliferation and represses myogenic differentiation by regulating Foxo4 and Mef2 factors

“…The mouse Foxk1 cDNA was inserted under the control of the cytomegalovirus (CMV) promoter/enhancer upstream of an ires-GFP fragment to produce a bicistronic pAC shuttle plasmid (Ivanciu et al, 2007). To construct a negative control, no cDNA was inserted into this vector.…”

Anti-inflammatory actions of serine protease inhibitors containing the Kunitz domain

“…Downregulation of TFPI-2 expression has been related to pathophysiological processes such as inflammation, angiogenesis, atherosclerosis and tumor growth/ metastasis [30,74,75]. As a consequence of decreased MMP-2 activation, TFPI reduced ECM degradation, tumor growth and metastasis [76,83]. Thus, understanding the protease specificity of each Kunitz domain is biologically significant.…”