Tissue Factor Pathway Inhibitor-2 Is Upregulated by Vascular Endothelial Growth Factor and Suppresses Growth Factor-Induced Proliferation of Endothelial Cells
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-1β, and modestly increased by both tumor necrosis factor (TNF)-α 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.
- cell signaling
- endothelial cells
- tissue factor pathway inhibitor-2
- vascular endothelial growth factor
Angiogenesis, the formation of new blood vessels from pre-existing ones, is a complex process including extracellular matrix breakdown, endothelial cell sprouting, proliferation, migration, and differentiation, and recruitment of pericytes. It is not only a natural physiological process but plays a critical role in a variety of pathological disorders such as diabetic retinopathy, age-related macular degeneration, and tumor progression.1–3 VEGF is known to be a critical pro-angiogenic factor with its ability to regulate many steps in the angiogenic process, including proliferation, migration, and survival.
TFPI-2 is a member of the Kunitz-type family of serine protease inhibitors, with strong homology to tissue factor pathway inhibitor (TFPI-1).4,5 Recombinant TFPI-2 exhibits strong inhibitory activity toward trypsin, factor XIa, plasma kallikrein, and plasmin, and weaker inhibitory activity for factor VIIa-tissue factor complex, factor IXa-polylysine, and cathepsin G.4,6 In addition, TFPI-2, as well as its first Kunitz-type domain, has been reported to inhibit matrix metalloproteinases,7,8 although a recent report failed to observe inhibition of activated matrix metalloproteinase (MMP)-1 by recombinant TFPI-2.9 TFPI-2 contains 3 tandem Kunitz-type domains, and the first Kunitz-type domain (KD1) plays an essential role in proteinase inhibition.10 In light of its properties, TFPI-2 has a possible role in the coagulation cascade4 and likely plays an important role in the regulation of extracellular matrix digestion and remodeling.11 In addition, TFPI-2 is becoming increasingly recognized as a regulatory molecule in tumor progression, with significant inhibitory effects on tumor growth and metastasis.11
TFPI-2 is constitutively synthesized and secreted by a variety of human endothelial cells and is deposited into the extracellular matrix of these cells.12 Its localization in the vascular endothelium of healthy human blood vessels suggests that it might play a physiological role in the healthy vascular wall.13 Recently, TFPI-2 has been shown to exhibit strong inhibitory effects on tumor angiogenesis.14 In these studies, recombinant adeno-associated virus (AAV)-mediated TFPI-2 expression in glioma cells reduced capillary-like structure formation by endothelial cell co-cultures in vitro and inhibited the formation of microvessels in vivo.14 One possible mechanism for the anti-angiogenic effect of TFPI-2 is the modulation of levels of angiogenic growth factors. Fibrosarcoma cells stably transfected with a TFPI-2 expression plasmid produced significantly smaller tumors compared with corresponding control fibrosarcoma cells. The tumors from the TFPI-2 transfected fibrosarcoma cells exhibited 3- to 6-fold lower levels of VEGF gene expression.15
The regulation of TFPI-2 gene expression in endothelial cells and the direct effects of TFPI-2 on endothelial cell function (particularly in the context of angiogenesis) have not been studied extensively. Here we investigated the expression of TFPI-2 and its upregulation by VEGF in retinal microvascular and umbilical vein endothelial cells. In addition, we investigated the ability of TFPI-2 to regulate VEGF-induced and FGF-2–induced endothelial cell activation. We found that TFPI-2 mRNA and protein expression in endothelial cells is significantly upregulated by VEGF in a MEK-dependent fashion. Furthermore, recombinant TFPI-2 suppresses both VEGF-induced proliferation and migration of endothelial cells, whereas recombinant KD1 inhibits VEGF-induced migration, but has no effect on VEGF-induced proliferation. Finally, TFPI-2 suppresses VEGF-induced activation of extracellular signal-regulated kinase (ERK) and Akt in endothelial cells. These results suggest that TFPI-2 may play an anti-angiogenic role with direct inhibitory effects on endothelial cells and may therefore represent a novel therapeutic target for angiogenic disease processes.
Materials and Methods
Reagents and Antibodies
Human recombinant VEGF, placental growth factor (PlGF), fibroblast factor (FGF)-2, tumor necrosis factor (TNF)-α, and IL-1β were purchased from R&D Systems (Minneapolis, Minn). U0126 and PD98059 were purchased from CalBiochem (San Diego, Calif). Other chemicals and reagents were obtained from Sigma Chemical Company (St. Louis, Mo) unless otherwise indicated. Recombinant human TFPI-2 and protein A-Sepharose–purified rabbit anti-human TFPI-2 IgG were prepared as previously described.10,12 Recombinant, His-tag–free preparations of human KD1, R24K KD1, and R24Q KD1 were expressed in Escherichia coli essentially as described using the pET28a plasmid (Invitrogen) containing a His6 leader sequence followed by a thrombin cleavage site and the cDNA encoding either wild-type KD1 or mutant KD1.16 Anti-phospho-VEGF receptor 2 antibodies (Tyr 1175), anti-phospho-p44/42 MAPK (Thr202/Tyr204) antibodies, anti-phospho-Akt (Ser473) antibodies, anti-p44/42 MAPK antibodies, and anti-Akt1 antibodies were all from Cell Signaling Technologies (Beverly, Mass). Anti-GAPDH antibodies were ordered from Abcam Inc (Cambridge, Mass).
Human retinal endothelial cells (HRECs) (Cell Systems, Kirkland, Wash) and human umbilical vein endothelial cells (HUVECs) (Clonetics, San Diego, Calif) were cultured in EGM2-MV medium (Clonetics) and used between passages 4 and 10. For the signaling pathway studies, confluent cells cultured in 6-well plates were serum-starved in OPTI-MEM plus 0.5% fetal bovine serum (FBS) overnight and pretreated with different doses of rh-TFPI-2 or KD1 preparations for 1 hour, then stimulated with 10 ng/mL VEGF for 10 minutes. For the inhibitor studies, serum-starved cells were pretreated with pharmaceutical inhibitors for 30 minutes.
Real-Time PCR Analysis
Cells were harvested in TRIZOL (Invitrogen), and total RNA was isolated according to manufacturer’s instructions. Single-stranded cDNA was synthesized from 1 μg total RNA using an oligo (dT) 18-mer as primer and the MMLV reverse transcriptase (Invitrogen) in a final reaction volume of 25 μL. Real-time PCR was performed with the QuantiTect SYBR Green PCR Kit (Qiagen, Valencia, Calif) and LightCycler system (Roche Diagnostics, GmbH, Germany). The primers were: hTFPI-2 sense 5′-GGGCCCTACTTCTCCGTTAC-3′ and antisense 5′-CACACTGGTCGTCCACACTC-3′. GAPDH was used as the reference for normalization, using hGAPDH sense primer 5′-GAGTCAACGGATTTGGTCGT-3′ and antisense 5′-GACAAGCTTCCCGTTCTCAG-3′. TFPI-2 values were normalized to GAPDH gene expression.
Western Blot Analysis
Total cell lysates were harvested in Laemmli sample buffer (BioRad Laboratories, Hercules, Calif). Extracellular matrix (ECM) preparations were obtained by lysing cells with Triton X-100 as previously described.17 Briefly, cells were washed with phosphate-buffered saline (PBS) 3 times and lysed by incubation with 0.5% Triton X-100 in PBS for 20 minutes at room temperature. After washing 3 times with PBS and another 3 times with 20 mmol/L Tris-HCl (pH 7.4) containing 100 μmol/L NaCl and 0.1% Tween-20, the ECM was collected in 200 μL Laemmli sample buffer. The protein samples from total cell lysates or ECM were separated by SDS-PAGE and transferred to Hybond ECL nitrocellulose membrane (Amersham Biosciences, Piscataway, NJ). After incubating with appropriate primary and secondary antibodies, the blots were detected with the Supersignal West Pico Chemiluminescent Substrate (Pierce Biotechnology, Rockford, Ill).
Endothelial cell proliferation activity was measured using the Quick Cell Proliferation Assay Kit (Biovision Inc, Mountain View, Calif). Briefly, 2.5×103 cells in EBM-2 supplemented with 2% FBS were added in quadruplicate to each well of 96-well plates and incubated at 37°C in 5% CO2. After 2 to 3 hours, the medium was replaced with 100 μL fresh EBM-2 with 2% FBS containing 10 ng/mL FGF-2 or 25 ng/mL VEGF plus different doses of rhTFPI-2 or various KD1 preparations. After 72 hours of incubation, 10 μL WST1/ECS solution from the kit was added to each well and incubated at 37°C in 5% CO2 for another 2 to 3 hours. The plates were shaken thoroughly, and the absorbance measured using a microplate reader (Synergy HT; BioTeK Instruments, Inc) at 450 nm and 630 nm.
Modified Boyden Chamber Migration Assay
Modified Boyden chambers containing polycarbonate membranes (Transwell, 8 μm pore size; Corning Inc) coated with 10 μg/mL collagen were used.18 Serum-starved HUVECs and HRECs were trypsinized and resuspended in EBM2 containing 0.1% bovine serum albumin. After preincubation with 1 μmol/L TFPI-2, wt-KD1, R24K-KD1, or R24Q-KD1 for 1 hour, the cells were seeded onto the upper chamber at 1×105 per insert. The lower chamber contained EBM2–0.1% bovine serum albumin plus VEGF and TFPI-2 or various KD1 preparations. After incubation at 37°C for 4 hours, the stationary cells on the top of the membrane were removed by a cotton swab and the migrated cells on the bottom of the membrane were stained with DAPI. Ten photographs for each well were randomly taken under microscope (Zeiss Axiovert 200) at 20× objective, and cells were counted using the Image J program.
Wound Healing Migration Assay
Confluent HUVECs on 24-well plates were serum-starved in EBM-2 plus 0.1% FBS overnight. The monolayer was scratched with a sterile 10-μL tip. The cells were gently washed with warm EBM2 to remove detached cells, preincubated with TFPI-2 or various KD1 domain preparations for 1 hour, and then stimulated with 10 ng/mL VEGF for 6 hours. Four injured fields in each well were photographed, and cell migration was quantified by measuring the recovered area using Image J program.
All experiments were performed at least 2 times. The results were reported as mean±SD. An unpaired Student t test was used to determine statistical significance. A value of P<0.05 was considered as significant.
TFPI-2 Expression Is Regulated by Growth Factors and Cytokines in Endothelial Cells
TFPI-2 has previously been found to be expressed by HUVECs19 and other endothelial cell types,12 and its expression in endothelial cells is regulated by inflammatory mediators including TNF-α. We were interested in determining whether TFPI-2 might be regulated by angiogenic growth factors, particularly VEGF. We performed real-time PCR analysis of HUVECs treated with VEGF, PlGF, FGF-2, TNF-α, or IL-1β. TFPI-2 mRNA expression was dramatically upregulated by both VEGF and IL-1β, and modestly increased by TNF-α and FGF-2. In contrast, PlGF did not induce the expression of TFPI-2 mRNA (Figure 1).
VEGF Upregulates TFPI-2 Expression in Human Endothelial Cells
We were interested in further characterizing the upregulation of TFPI-2 in endothelial cells by VEGF. We performed real-time PCR and Western blot analysis in HRECs and HUVECs to investigate VEGF regulation of TFPI-2 expression. As shown in Figure 2A, 25 ng/mL VEGF increased TFPI-2 expression by 4.7-fold after 4 hours of treatment. TFPI-2 mRNA expression was maximal at 24 hours and 12 hours in HUVECs and HRECS, respectively. In contrast, TFPI-1 mRNA levels were not increased, and actually decreased, when endothelial cells were treated with VEGF (data not shown). Western blot analysis for TFPI-2 protein expression in HUVECs indicated increased protein expression consistent with increased levels of TFPI-2 transcript (Figure 2B). We detected 3 different glycosylated isoforms of TFPI-2 in extracellular matrix preparations, in agreement with previous observations,12,19 and all 3 isoforms were upregulated by VEGF treatment in HUVEC. VEGF elevated TFPI-2 expression in a dose-dependent manner, with a peak effect at 25 to 100 ng VEGF/mL (Figure 2C).
MEK Pathway Is Involved in VEGF-Increased TFPI-2 Expression
We next investigated the role of the MEK pathway in VEGF-induced TFPI-2 upregulation. As shown in Figure 3, the MEK1/2 inhibitor U0126 almost completely abrogated the VEGF-induced TFPI-2 upregulation. The MEK1 inhibitor PD98059 also significantly inhibited the VEGF induction of TFPI-2 expression. Taken together, these results demonstrate that VEGF-induced TFPI-2 expression in endothelial cells is dependent on MEK signaling.
TFPI-2 Inhibits Growth Factor Stimulation of Endothelial Cell Proliferation
Virus-mediated TFPI-2 expression in glioma cells was previously demonstrated to inhibit capillary-like structure formation by endothelial cell co-cultures in vitro as well as the formation of microvessels in vivo.14 Because TFPI-2 is upregulated in endothelial cells by VEGF, we were interested in determining whether TFPI-2 regulates EC activities such as proliferation and migration. As shown in Figure 4A, TFPI-2 at concentrations of 0.5, 1, and 2 μmol/L inhibited 25 ng/mL VEGF-induced proliferation by 60%, 85%, and 85% in HUVECs. In contrast, a recombinant preparation of wild-type KD1, which contains all the proteinase inhibitory activity of TFPI-2, did not affect VEGF-induced proliferation in HUVECs, even at a concentration of 2 μmol/L. TFPI-2 also suppressed FGF-2-stimulation of HUVEC proliferation in a dose-dependent fashion (74% inhibition at 2 μmol/L), whereas wt-KD1 had no effect on FGF-2–induced proliferation (Figure 4B). We found very similar results in HRECs (data not shown).
Effect of TFPI-2 on VEGF-Induced Endothelial Cell Migration
In addition to endothelial cell proliferation, we also investigated whether TFPI-2 modulates EC migration. Both a modified Boyden chamber assay and the wound healing assay were performed to investigate whether TFPI-2 had any effect on VEGF-induced migration in endothelial cells. As shown in Figure 5A and 5B, 10 ng/mL VEGF increased migration of HUVEC by 300% in the Boyden chamber experiment. Addition of 1 μmol/L TFPI-2 significantly suppressed VEGF-induced migration by 40%. An equivalent concentration of wt-KD1 and R24K-KD1 (which has increased inhibitory activity toward both plasmin and trypsin10) suppressed VEGF-induced migration by 74% and 82%, respectively. R24Q-KD1, which exhibits ≈10% of the inhibitory activity toward trypsin and plasmin as compared with KD1,20 had no significant inhibitory effect on migration. Similar results were obtained in the wound healing experiments, as shown in Figure 5C and 5D. TFPI-2, wt-KD1, and R24K-KD1 showed significant inhibitory activities on VEGF-induced migration, whereas R24Q-KD1 had no significant effect on migration.
TFPI-2 Blocks VEGF-Induced Activation of Akt and ERK in Endothelial Cells
Because TFPI-2 blocked VEGF stimulation of EC proliferation, we were interested in determining whether TFPI-2 inhibited VEGF induction of signaling pathways, particularly ERK1/2 and Akt. VEGF (10 ng/mL) strongly activated the phosphorylation of both ERK and Akt in HUVECs (Figure 6A). Preincubation of HUVECs with TFPI-2 significantly inhibited VEGF-induced ERK and Akt phosphorylation in a dose-dependent manner (Figure 6A). In contrast, wt-KD1, even at a concentration of 2 μmol/L, did not affect VEGF-induced phosphorylation of ERK and Akt (Figure 6B).
TFPI-2 Suppresses VEGF-Induced Tyrosine Phosphorylation of VEGF Receptor-2 in HUVECs
Because TFPI-2 inhibits VEGF-induced ERK and Akt signaling, it was of interest to determine whether TFPI-2 regulates VEGF-induced activation of VEGFR2. Treatment of HUVECs with VEGF (10 ng/mL) strongly stimulated the tyrosine phosphorylation of VEGFR-2, as expected (Figure 6C). Preincubation of HUVECs with TFPI-2 at a concentration of 2 μmol/L significantly suppressed VEGF-induced phosphorylation of VEGFR2; lower concentrations of TFPI-2 (0.5 and 1 μmol/L) suppressed phosphorylation of VEGFR2 to a significantly lesser extent (Figure 6C).
TFPI-2 is secreted by a variety of human endothelial cells,12 and its localization in the vascular endothelium of human blood vessels suggests that it may play a physiological role in the healthy vascular wall.13 In addition, viral mediated gene transfer of TFPI-2 has been shown to exhibit strong inhibitory effects on tumor angiogenesis.14 We were therefore interested in learning more about the expression and activity of TFPI-2 in endothelial cells.
TFPI-2 gene expression in endothelial cells is known to be upregulated by the inflammatory mediators lipopolysaccharide and TNF-α.12 TFPI-2 expression is increased in the mouse liver during the acute phase reaction, and this increase is thought to occur in the endothelial cells.21 While our studies demonstrated a mild upregulation of TFPI-2 expression in endothelial cells by TNF-α, we found that IL-1β dramatically increased (>10-fold) TFPI-2 expression. It is quite possible, therefore, that IL-1β is also an important mediator of TFPI-2 upregulation in endothelial cells during acute inflammation.
Of particular interest for angiogenesis, we found that VEGF also dramatically increased EC expression of TFPI-2 mRNA and protein, whereas FGF-2, by comparison, mildly increased TFPI-2 expression. The VEGF-mediated induction of TFPI-2 gene expression in endothelial cells was strongly dependent on MEK1/2, the kinase responsible for ERK1/2 activation. This utilization of ERK in the upregulation of TFPI-2 in endothelial cells is consistent with ERK-dependent TFPI-2 gene regulation in other cell types.22,23
VEGF upregulation of TFPI-2 expression in ECs is intriguing in light of the possible anti-angiogenic effects of TFPI-2. Viral mediated TFPI-2 gene transfer in glioma cells inhibits the formation of microvessels in vivo.14 One possible mechanism for the anti-angiogenic activity of TFPI-2 is the modulation of growth factor levels.15 Our studies indicate that TFPI-2 also has direct anti-angiogenic effects on endothelial cells, because TFPI-2 significantly suppresses both VEGF-stimulated and FGF-2–stimulated endothelial cell proliferation. In contrast to TFPI-2, a recombinant preparation of wild-type KD1, which contains all the proteinase inhibitory activity of TFPI-2, did not affect growth factor-induced proliferation in HUVECs. This suggests that the anti-proliferative activity of TFPI-2 involves a mechanism separate from its proteinase-inhibitory activity.
However, recombinant TFPI-2 and wild-type KD1 significantly suppressed VEGF-induced EC migration in 2 separate assays. In addition, the R24K mutant of KD1, which exhibits an increased inhibitory activity toward both plasmin and trypsin (Ki=0.85 nM for plasmin20), suppressed VEGF-induced migration more efficiently than TFPI-2 and wild-type KD1 (Ki values for the inhibition of plasmin of 9 nM and 3 nM, respectively20). In contrast, the R24Q mutant of KD1, which exhibits substantially reduced inhibitory activity toward trypsin and plasmin20 (Ki=48 nM for plasmin20), had no significant inhibitory effect on migration. These results strongly suggest that the inhibitory effect of TFPI-2 on VEGF-induced migration is mainly dependent on its serine proteinase inhibitory activity.
VEGFR2 is considered to be the major mediator of several physiological and pathological effects of VEGF on endothelial cells, including mitogenesis and survival.24 Two of the major signaling pathways stimulated by VEGF in endothelial cells are the Raf-1/MEK/ERK cascade and the PI3-kinase/Akt pathway.25 Our studies indicate that TFPI-2, but not KD1, suppresses VEGF-induced activation of both Akt and ERK in a dose-dependent fashion. Because ERK signaling is important in VEGF-induced proliferation in endothelial cells,25 suppression of ERK is a plausible mechanism for TFPI-2 inhibition of EC proliferation, although other mechanisms may also be contributory.
Because TFPI-2 suppresses VEGF activation of both ERK and Akt, an obvious question was whether TFPI-2 suppresses VEGF activation of VEGFR2/KDR. Our studies indicate that TFPI-2 at higher doses of 2 μmol/L significantly suppresses VEGF activation of KDR, whereas lower doses of TFPI-2 resulted in, at best, modest suppression (Figure 6C). In contrast, TFPI-2 at doses of 0.5 μmol/L significantly suppressed VEGF activation of both ERK and Akt (Figure 6). These results provide suggestive evidence that TFPI-2 can suppress ERK and Akt stimulation by multiple mechanisms, including suppression of VEGFR2 stimulation.
In light of the anti-angiogenic properties of TFPI-2, it is interesting that VEGF strongly induces endothelial cell gene expression of this molecule. Angiogenesis involves an interplay of both stimulatory and inhibitory factors to prevent excessive angiogenesis and unchecked endothelial cell activation. VEGF activation of endothelial TFPI-2 expression could represent a physiological negative feedback regulatory mechanism, similar to the recently characterized vasohibin molecule.26,27 It has previously been demonstrated that the majority of the TFPI-2 secreted by endothelial cells is deposited into their extracellular matrix.12 Based on an analysis of absolute TFPI-2 quantities extracted from the ECM of HUVECs12 and an ECM thickness28 of 30 to 100 microns, we estimate a potential TFPI-2 concentration of 0.008 to 0.024 μmol/L in the ECM of unstimulated HUVECs and 0.08 to 0.24 μmol/L in VEGF-stimulated HUVECs (the potential concentration could be even higher in other endothelial cell types12). This indicates that induction of TFPI-2 by VEGF could play a functional role in modulating VEGF’s angiogenic activity. In this regard, we attempted to address this hypothesis using either polyclonal12 or monoclonal15 anti-TFPI-2 antibody preparations. In our studies, each anti-TFPI-2 McAb (SK-2, SK-8 and SK-9) failed to neutralize the anti-angiogenic/anti-proliferative effect of TFPI-2. In addition, the protein A-Sepharose–purified rabbit anti-TFPI-2 IgG preparation12 also failed to neutralize this activity, presumably because of the fact that the “specific” anti-TFPI-2 IgG represented ≈10% of the total IgG concentration, thus resulting in our inability to achieve a molar concentration of polyvalent anti-TFPI-2 IgG in excess of TFPI-2. Accordingly, the role of TFPI-2 as a feedback inhibitor of VEGF action remains speculative. Further studies are warranted to define the role of endogenous TFPI-2 in VEGF action and to determine the therapeutic potential of TFPI-2 or its derivatives in the treatment of angiogenic disease states.
Sources of Funding
This work was supported by research grants from the National Institutes of Health (EY00398, EY014136 and HL64119), the Juvenile Diabetes Research Foundation, Knights Templar Eye Foundation, Mary Kathryn and Michael B. Panitch Fund, and Richard and Sandra Forsythe/Forsythe Technology, Inc.
Original received April 26, 2006; final version accepted September 22, 2006.
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