Vascular Endothelial Cell Growth Factor Attenuates Actions of Transforming Growth Factor-β in Human Endothelial Cells*

Because vascular endothelial cell growth factor (VEGF) and transforming growth factor-β (TGF-β) are both involved in cellular growth and differentiation, we examined whether VEGF modifies TGF-β signaling cascade in human umbilical cord vein endothelial cells (HUVEC). Production of plasminogen activator inhibitor-1 (PAI-1), which is under the specific control of TGF-β, was strongly enhanced (3.5-fold) by TGF-β treatment. Remarkably, physiological concentration of VEGF (30 nm) profoundly (by 60%) attenuated the TGF-β stimulation of PAI-1 production without an effect on the basal PAI-1 production. In HUVECs transiently transfected with an expression construct containing a PAI-1 promoter fused to luciferase reporter gene, TGF-β-stimulation of transcription of PAI-1 was clearly (by 60%) inhibited by VEGF. TGF-β phosphorylation of Smad2/3, an obligatory step of intracellular TGF-β signaling, was also suppressed by VEGF. VEGF attenuation of TGF-β action was also demonstrated in two other endothelial cell lines. In conclusion, VEGF attenuates TGF-β action in the human endothelial cell, specifically at the level of transcription of PAI-1 gene and Smad2/3 phosphorylation.

Peptide growth factors are implicated in the development and progression of pathological changes of blood vessels (1,2). The involvement of vascular endothelial cell growth factor (VEGF) 1 in these processes has been investigated extensively (3,4). VEGF plays a major part in mediating active intraocular neovascularization in patients with ischemic retinal diseases such as diabetic retinopathy (5). A role of VEGF in the development of nonproliferative diabetic retinopathy was also reported (6). On the other hand, transforming growth factor-␤ (TGF-␤), a strong modulator of cell growth and differentiation, exhibits either deleterious or protective effects on the blood vessels depending on the experimental conditions (7,8). Considering that many growth factors are simultaneously acting on the same tissue in vivo, delineation of synergistic and/or antagonizing effects of growth factors is important for understanding of pathophysiology of diabetic complications (9 -11).
Accordingly, we systematically analyzed an interaction between VEGF and TGF-␤ in the endothelial cell lines, and discovered a novel action of VEGF, the attenuation of TGF-␤ action.
Plasmid Constructs-A DNA of the genomic sequence from nucleotides Ϫ799 to ϩ71 of the 5Ј end of the human plasminogen activator inhibitor-1 (PAI-1) gene was inserted into the thymidine kinase-luciferase, which contains the herpes simplex virus thymidine kinase minimal promoter driving expression of the luciferase reporter gene (PAI-1/luciferase) (14). This promoter fragment was specifically regulated by TGF-␤ (14). This plasmid was a generous gift from Dr. Mayumi Abe (Tohoku University, Tohoku, Japan).
Cell Culture and Transfection-A human umbilical vein endothelial cell line (HUVEC) (Japanese Collection of Research Bioresources Cell Bank, Osaka, Japan), a cultured human dermal microvascular endothelial cell line (CADMEC) (Kurabo Industries, Osaka), and a bovine coronary artery endothelial cell line (BCAEC) (Kurabo Industries, Osaka) were maintained in human endothelial serum-free medium (Invitrogen) containing 20 ng/ml basal fibroblast growth factor and 10 ng/ml epidermal growth factor on a collagen I-coated dish. PAI-1/luciferase was transfected into HUVEC using an electroporation method. Briefly, HUVEC were electroporated with a total of 10 g of plasmid DNA at 250 V and 960 microfarad. After the electroporation, the cells were plated to a 24-well dish in Dulbecco's modified Eagle's medium containing 10% heat-inactivated fetal bovine serum. Twelve hours later, the incubation medium was changed to serum-free F-12 medium containing test substances. After the 12-h test incubation, the media and the cells were collected for determination of PAI-1 and luciferase activity, respectively. TGF-␤ and/or VEGF treatment did not significantly change the cell number and protein/cell under the experimental conditions. All experiments were performed at least three times in triplicate.
PAI-1 Assay-PAI-1 concentration in the medium was determined using a commercially available enzyme-linked immunosorbent assay kit (Biopool International, Ventura, CA).
Luciferase Assay-The cells were lysed using Promega lysis buffer (Promega Corp., Madison, WI), mixed with the Promega luciferase assay reagent, and the luciferase activity of the cells was detected by luminometer. To correct for differences in transfection efficiencies between plates within an experiment, the luciferase activity in an extract was normalized to the ␤-galactosidase activity unless otherwise indicated.
Immunoblotting-Whole cell extracts were prepared by detergent solubilization of the cells in the lysis buffer (20 mM Hepes, pH 7.4, 1% Triton X-100, 2 mM EDTA, 100 mM sodium fluoride, 10 mM sodium pyrophosphate, 2 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 10 M leupeptin, 10 g/ml aprotinin, and 1.5 M pepstatin) * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
for 1 h at 4°C. For Western blotting, 200 g of protein lysate was applied for each lane. Two mg of protein lysate was diluted five times by lysis buffer without Triton X-100 and used for immunoprecipitation experiments in which the diluted cell lysate was incubated with 2 g of aSmad7 antibody for 4 h at 4°C. The primary polyclonal antibody was incubated with protein A-agarose for 2 h at 4°C. The resultant cell extracts or immunoprecipitates were subjected to SDS-polyacrylamide gel electrophoresis and Western blotting using Tris-buffered saline with Tween 20 including 3% bovine serum albumin for blocking solution and 1000ϫ diluted primary antibody of apSmad, aSmad2/3, aTGFIR, or aSmad7 and visualized with the enhanced chemical luminescence detection system (ECL, Amersham Biosciences). The densitometry data were evaluated by NIH Image, version 1.63 (Bethesda, MD).
Statistical Analysis-Analysis of variance with Fisher's protected least significant difference test (StatView, SAS Institute Inc., Cary, NC) was employed for statistical analysis, and p Ͻ 0.05 was considered significant.

Stimulation of PAI-1 production by TGF-␤ in HUVEC and Its
Suppression by VEGF-Accumulation of PAI-1 in the culture medium during a 12-h incubation period was used as an index of PAI-1 production. One nM TGF-␤ increased PAI-1 production 3.5-fold, and 30 nM VEGF significantly (60%) inhibited such TGF-␤ action (Fig. 1). Nevertheless, VEGF did not significantly change basal PAI-1 production in the absence of TGF-␤.
Stimulation of PAI-1 Gene Transcription by TGF-␤ in HUVEC and Its Attenuation by VEGF-In HUVEC cells trans- fected with PAI-1/luciferase, the effects of TGF-␤ and VEGF on PAI-1 promoter-induced luciferase activity were examined. As expected, TGF-␤ stimulated luciferase activity; the EC 50 was 30 pM, and the maximum effect was at 100 pM (Fig. 2), indicat-ing the validity of this cell line. VEGF, when added alone, did not cause a significant change in PAI-1 promoter activity; however, the peptide strongly inhibited the TGF-␤ stimulation of PAI-1 promoter activity in a dose-dependent manner (Fig. 3). The IC 50 of VEGF effect was 3 nM, and the maximum inhibition occurred at 30 nM. Importantly, VEGF attenuated TGF-␤ stimulation of PAI-1 promoter activity also in CADMEC and BCAEC (Fig. 4). VEGF121 showed a similar effect at 30 nM (Fig. 5A).
The data imply that VEGF inhibits TGF-␤-induced activation of PAI-1 gene at the level of transcription, leading to a reduced PAI-1 production in endothelial cells.
Effect of VEGF on Association of TGF-␤ Receptor and Smad7-Finally we estimated expression and binding to the TGF-␤ receptor of Smad7, a negative modulator of TGF-␤ signaling. Pretreatment with 30 nM VEGF increased the binding of Smad7 (by 65%) to the TGF-␤ receptor (Fig. 7). As the treatment did not alter the amount of Smad7 and TGF-␤ receptor, the finding indicated that VEGF enhances the binding of Smad7 to the TGF-␤ receptor. DISCUSSION TGF-␤ potently stimulates PAI-1 production by endothelial cells (12,15,16), and induces angiogenesis (17). We found that VEGF itself did not significantly change PAI-1 production and PAI-1 promoter activity, namely mRNA expression. Quite interestingly, however, the addition of VEGF strongly inhibited TGF-␤ stimulation of PAI-1 production and mRNA expression. The data imply that VEGF inhibits TGF-␤-induced activation of the PAI-1 gene at the level of transcription, leading to a reduced PAI-1 production. PI3-kinase is one of the major intra- cellular signaling molecules for VEGF (18), and wortmannin, a potent PI3-kinase inhibitor, prevented the inhibitory effect of VEGF on TGF-␤-induced PAI-1 expression. Thus, inhibition of TGF-␤ action by VEGF is likely mediated by PI3-kinase.
There are three signaling tyrosine kinase receptors (VEGFR-1, VEGFR-2, and VEGFR-3) for three VEGFs (a fulllength VEGF, VEGF121, and VEGF-B). VEGFR-1 and VEGFR-2 are abundantly expressed in the bovine pulmonary artery cells (19). We found that not only a full-length VEGF (a ligand for the three VEGFRs) but also VEGF121 (a ligand for VEGFR-1 and VEGFR-2) suppressed TGF-␤ action. On the other hand, Olofsson et al. (20) showed that VEGF-B (a selective ligand for VEGFR-1) increases PAI-1 gene expression without PI3-kinase activation and that a full-length VEGF fails to increase PAI-1 activity in HUVEC. Taken together, VEGF may suppress PAI-1 activity via VEGFR-2-mediated activation of PI3-kinase.
Smad cascade is the major signaling pathway of TGF-␤ activation of PAI-1 expression (21,22). This occurs through ALK5/Smad2 (23). Because we did not examine the effect of VEGF on the other branch of TGF-␤ signaling (ALK1/Smad1), it is not known whether VEGF inhibits both branches of TGF-␤ signaling. TGF type I receptor mediates phosphorylation of Smad2/3 at the C-terminal serine residues, which is a required step for Smad signaling (13). Upon phosphorylation, Smad2 and Smad3 homo-or heterooligomerize with Smad4, and translocate into the nucleus (24 -26). There they bind to DNA and regulate gene transcription. The interaction of Smad2/3 with coactivators such as CBP and p300 is essential for TGF-␤ inhibition of cellular proliferation, and such interaction is enhanced by the phosphorylation of Smad2/3 by TGF-␤ (27). The cross-talk between epidermal growth factor and TGF-␤ signaling has also been reported (28,29).
As shown, TGF-␤ stimulation of phosphorylation of Smad2/3 was strongly suppressed by VEGF without an effect on the amount of TGF-␤ receptor. Therefore, VEGF must have inhibited TGF-␤ action through interference of the intracellular signaling. VEGF stimulation of Smad7 (an antagonist of TGF-␤ signaling) association with TGF-␤ type 1 receptor may well be responsible for such VEGF action.
In summary, we have discovered a novel action of VEGF in endothelial cells, the inhibition of TGF-␤ stimulation of PAI-1 gene activation and PAI-1 production. We suggest the following scenario: VEGF binding to VEGFR-2 activates PI3-kinase, leading to enhanced binding of Smad7 to TGF-␤ type 1 receptor and interference of TGF-␤-induced phosphorylation of Smad2/3, which is responsible for attenuation of TGF-␤ action. FIG. 7. Effect of VEGF on binding of Smad7 to TGF-␤ receptor type I in HUVEC. HUVEC cells were incubated with 30 nM VEGF for 60 min, and the cell lysates were immunoprecipitated (IP) by Smad7 antibody (aSmad7) and immunoblotted (IB) with antibodies against TGF-␤ receptor type I (aTGFRI) or aSmad7. These data are representative of three independent experiments. The densitometry data are presented as relative density compared with the density at the basal (untreated) condition. **, p Ͻ 0.01 versus basal expression.