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[Cancer Research 65, 1414-1424, February 15, 2005]
© 2005 American Association for Cancer Research

Cell and Tumor Biology

The Homeobox Gene Gax Inhibits Angiogenesis through Inhibition of Nuclear Factor-{kappa}B–Dependent Endothelial Cell Gene Expression

Sejal Patel, Alejandro D. Leal and David H. Gorski

Division of Surgical Oncology, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, The Cancer Institute of New Jersey, New Brunswick, New Jersey

Requests for reprints: David H. Gorski, Division of Surgical Oncology, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, The Cancer Institute of New Jersey, 195 Little Albany St., New Brunswick, NJ 08901. Phone: 732-235-8524; Fax: 732-235-8098; E-mail: gorskidh{at}umdnj.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The growth and metastasis of tumors are heavily dependent on angiogenesis, but much of the transcriptional regulation of vascular endothelial cell gene expression responsible for angiogenesis remains to be elucidated. The homeobox gene Gax is expressed in vascular endothelial cells and inhibits proliferation and tube formation in vitro. We hypothesized that Gax is a negative transcriptional regulator of the endothelial cell angiogenic phenotype and studied its regulation and activity in vascular endothelial cells. Several proangiogenic factors caused a rapid down-regulation of Gax mRNA in human vascular endothelial cells, as did conditioned media from breast cancer cell lines. In addition, Gax expression using a replication-deficient adenoviral vector inhibited human umbilical vein endothelial cell migration toward proangiogenic factors in vitro and inhibited angiogenesis in vivo in Matrigel plugs. To identify putative downstream targets of Gax, we examined changes in global gene expression in endothelial cells due to Gax activity. Gax expression resulted in changes in global gene expression consistent with a quiescent, nonangiogenic phenotype, with increased expression of cyclin kinase inhibitors and decreased expression of genes implicated in endothelial cell activation and angiogenesis. Further analysis revealed that Gax down-regulated numerous nuclear factor-{kappa}B (NF-{kappa}B) target genes and decreased the binding of NF-{kappa}B to its target sequence in electrophoretic mobility shift assays. To our knowledge, Gax is the first homeobox gene described that inhibits NF-{kappa}B activity in vascular endothelial cells. Because NF-{kappa}B has been implicated in endothelial cell activation and angiogenesis, the down-regulation of NF-{kappa}B–dependent genes by Gax suggests one potential mechanism by which Gax inhibits the angiogenic phenotype.

Key Words: Gax • homeobox genes • angiogenesis • vascular endothelium • NF-{kappa}B • transcription factors


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The process of angiogenesis, critical in both normal physiology and in disease states such as cancer and inflammatory diseases, is normally tightly regulated by a balance between pro- and antiangiogenic factors, known as the "angiogenic balance" (1). Tumors manipulate their microenvironment and parasitize the host by secreting factors that induce angiogenesis, tipping the angiogenic balance toward a proangiogenic state. The primary target of tumor-secreted proangiogenic factors is the vascular endothelial cell, which becomes "activated" and undergoes distinct changes in phenotype and gene expression. These changes include activation of proteolytic enzymes to degrade basement membrane, sprouting, proliferation, tube formation, and production of extracellular matrix (2, 3). Although the endothelial cell receptors and signaling pathways activated by proangiogenic factors such as vascular endothelial growth factor (VEGF; ref. 4) have been extensively studied, less is known about the molecular biology of the downstream transcription factors activated by these factors. Nuclear transcription factors likely integrate these upstream signals, activating and repressing downstream batteries of genes, to produce an angiogenic global gene expression profile, resulting in the angiogenic phenotype. Consequently, understanding the transcriptional mechanisms by which endothelial cells become activated is likely to suggest new therapeutic strategies for inhibiting this process at a very distal point in its signaling cascade, with potential applications in the antiangiogenic therapy of cancer.

Because of their ubiquitous role as regulators of cellular differentiation and body plan formation during embryogenesis, as well as oncogenes and tumor suppressors in various human cancers (5, 6), it is not surprising that homeobox genes have been implicated in regulating the phenotypic changes that endothelial cells undergo during angiogenesis (7). In particular, one diverged homeobox gene, Gax (whose mouse homologue is known as Meox-2), has several characteristics that suggest that it may play an important role as an inhibitor of the endothelial cell phenotypic changes that occur in response to stimulation by proangiogenic or proinflammatory factors (8–11). Originally isolated from vascular smooth muscle (8) and widely expressed in mesoderm and muscle precursors in the embryo (12, 13), in the adult Gax expression is mostly restricted to the cardiovascular system and kidney (8, 13). In vascular smooth muscle cells, Gax expression is down-regulated by mitogens and up-regulated by growth arrest signals (8, 14). Consistent with this observation, Gax expression induces G1 cell cycle arrest (10) and inhibits vascular smooth muscle cell migration, modulating integrin expression (11). In vivo, Gax expression in arteries inhibits proliferative restenosis of the arterial lumen after injury (10). Recently, we have reported that Gax is also expressed in endothelial cells, in which its expression inhibits endothelial cell proliferation (15) and strongly inhibits VEGF-induced endothelial cell tube formation on reconstituted basement membrane in vitro (15), suggesting that Gax may be an inhibitor of the activated, angiogenic phenotype.

Until now, we had not identified potential mechanisms by which Gax might accomplish its inhibition of endothelial cell activation, other than a general cell cycle arrest due to induction of p21 (10, 15). In this report, we now describe how Gax expression is regulated in endothelial cells by proangiogenic and proinflammatory factors and how its expression in endothelial cells can block angiogenesis in vivo. Finally, we present evidence that Gax inhibits nuclear factor-{kappa}B (NF-{kappa}B) activity in endothelial cells. Given that there is now considerable evidence that activation of NF-{kappa}B activity in endothelial cells is proangiogenic (16–22), this interaction between a homeobox gene and NF-{kappa}B represents one potential mechanism by which Gax expression may inhibit angiogenesis. This interaction, to our knowledge the first described in endothelial cells, may represent a new mechanism by which homeobox genes can interact with intracellular signaling pathways in endothelial cells and thereby inhibit tumor-induced angiogenesis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Lines and Expression Constructs
Human umbilical vein endothelial cells (HUVEC) and EGM-2 medium were obtained from BioWhittaker (Walkersville, MD) and HUVECs cultured according to the manufacturer's instructions. Human microvascular endothelial cells (HMEC)-1 cells were obtained from the Centers for Disease Control and were cultured as described (23). Breast cancer cell lines were obtained from the American Type Culture Collection (Manassas, VA) and cultured according to instructions. Conditioned medium was obtained by incubating them in serum-free medium for 24 hours.

The cloning of the Gax cDNA into the mammalian expression vector pCGN to produce pCGN-Gax and the construction of replication-deficient adenoviral vectors expressing the rat and human homologues of Gax (Ad.hGax and Ad.rGax, respectively) conjugated to the {alpha}-hemagglutinin epitope have been described (10). The control replication-deficient adenoviral vector expressing green fluorescent protein (Ad.GFP) was a kind gift of Dr. Daniel Medina (The Cancer Institute of New Jersey, New Brunswick, NJ). An adenoviral construct expressing a form of Akt (T308A, S473A, adenoviral construct designated Ad.DN.Akt) that functions as a dominant negative (24) was kindly provided by Dr. Kenneth Walsh (Boston University, Boston, MA). Expression of Gax protein was verified as previously described (13) by Western blot using antihemagglutinin antibody and anti-Gax antibodies (not shown). Transfections of HUVECs with pCGN-Gax were carried out using Trans-IT Jurkat transfection reagent (Mirus Bio Corporation, Madison, WI) according to a modification of the manufacturer's instructions.

Real-time Quantitative Reverse Transcription–PCR
After treatment as described individually for each experiment, total RNA was isolated from endothelial cells using a spin column with on-column DNase digestion to remove contaminating genomic DNA (RNAeasy, Qiagen, Valencia, CA). First-strand synthesis was done on the total RNA using oligo(dT) primers (SuperScript kit, Invitrogen, Carlsbad, CA), and then message levels for Gax and other genes determined by real time quantitative reverse transcription–PCR (RT-PCR) using TaqMan probes (25). Quantitative RT-PCR was carried out using a Cepheid SmartCycler thermocycler, with the associated SmartCycler v.2.0 software used to analyze the data and determine the threshold count (Ct).

Primer and probe sets for each gene were designed using the MacVector 7.2 software package (Accelrys, San Diego, CA). The fluorophore used was 6-carboxyfluorescein (6-FAM), and the quencher was Black Hole Quencher-1 (BHQ-1, Biosearch Technologies, Novato, CA). Sequences of the primers and probes were as follows: Gax: 5'-TCA GAA GTC AAC AGC AAA CCC AG-3' (forward), 5'-CCA GTT CCT TTT CCC GAG-3' (reverse), 5'-(6-FAM)-TGG TTC CAA AAC AGG CGG ATG-3'-(BHQ1; TaqMan probe), amplicon = 238 bp; E-selectin: 5'-CTC TGA CAG AAG AAG CCA AG-3' (forward), 5'-ACT TGA GTC CAC TGA AGT CA-3' (reverse), 5'-(6-FAM)-CCA CGC AGT CCT CAT CTT TTT G-3' (BHQ1; TaqMan probe), amplicon = 255 bp; vascular cell adhesion molecule-1 (VCAM-1): 5'-ATG ACA TGC TTG AGC CAG G-3' (forward), 5'-GTG TCT CCT TCT TTG ACA CT-3' (reverse), 5'-(6-FAM)-CAC TTC CTT TCT GCT TCT TCC AGC-3' (BHQ1; TaqMan probe), amplicon = 260 bp; intercellular adhesion molecule-1 (ICAM-1): 5'-TAT GGC AAC GAC TCC TTC T-3' (forward), 5'-CAT TCA GCG TCA CCT TGG-3' (reverse), 5'-(6-FAM)-CCT TCT GAG ACC TCT GGC TTC G-3'-(BHQ1; TaqMan probe), amplicon = 238 bp; GRO-{alpha}: 5'-CAA GAA CAT CCA AAG TGT GAA CG-3' (forward), 5'-(6-FAM)-AGG AAC AGC CAC CAG TGA GC-3' (reverse), 5'-CGC CCA AAC CGA AGT CAT AGC-3'-(BHQ-1; TaqMan probe), amplicon=200 bp. Sequences of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primer and probe set were 5'-ACA ACT TTG GTA TCG TGG AAG-3' (forward), 5'-CAG ATG AGG CAG GGA TGA TGT TC-3' (reverse), and 5'-(6-FAM)-ACC CAG AAG ACT GTG GAT GG-3'-(BHQ1; TaqMan probe), amplicon = 138 bp. For some experiments (Fig. 1), a set of primers for human Gax and ß-actin previously described were used (15), along with SYBr Green to monitor the PCR reaction.



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  		Figure 1. Gax expression is down-regulated induced in HUVECs by serum and up-regulated when serum is withdrawn. Using real-time quantitative RT-PCR, Gax levels were measured in quiescent HUVECs stimulated with serum and randomly cycling HUVECs placed in low-serum medium. Gax levels were normalized to ß-actin. For this experiment alone, primers for Gax and ß-actin previously described were used (15). Similar results were obtained with the primer/probe combination described in Materials and Methods. A, Gax is down-regulated by serum. B, Gax is up-regulated by serum withdrawal. C, PCR gel of the experiment in A. Units are arbitrary.

 
Real-time PCR cycles started with an initial 1.5-minute denaturation step at 95°C, followed by 30 to 40 cycles of denaturation at 95°C for 10 seconds; annealing at 50°C (VCAM-1), 52°C (E-selectin, ICAM-1), and 56°C (Gax, GAPDH, p21, Gro-{alpha}) for 20 seconds; and extension at 72°C for 30 seconds. Each sample was run in triplicate and Ct determined for the target gene. For all reactions, negative controls were run with no template present, and random RNA preparations were also subjected to sham quantitative RT-PCR (no reverse transcriptase) to verify lack of genomic DNA contamination. To correct for differences in RNA quality and quantity between samples, target gene levels were normalized to corresponding GAPDH message levels using the {Delta}{Delta}Ct method (26), as described previously (27, 28).

Migration Assays
Before each experiment, cell culture membranes and flasks were coated with sterile 0.1% gelatin in PBS. HUVECs were infected with adenoviral vectors for 16 hours before 5 x 104 cells per well were plated onto 8.0-µm pore size polycarbonate membrane in 24-well plates. Cells were allowed to attach for 1 hour in EGM-2 medium. Once the cells had attached, the medium in the upper chamber was replaced with low-serum medium [which consisted of EGM-2 + 0.1% fetal bovine serum (FBS) lacking VEGF, basic fibroblast growth factor (bFGF), and epidermal growth factor], and the lower chamber with low-serum medium supplemented with either 50 ng/mL VEGF, 50 ng/mL bFGF, 15 ng/mL tumor necrosis factor (TNF), or 10% FBS. VEGF, bFGF, and TNF-{alpha} all obtained from R&D Systems (Minneapolis, MN). After 5 hours, the inserts were washed with PBS and the upper surfaces cleaned with a cotton swab to remove any cells that had not migrated. Finally the cells were fixed with Diff-Quik Stain (Dade Behring, Deerfield, IL) and the inserts washed in PBS and photographed for counting. Cells were counted in five high-powered fields per well. Experiments were repeated at least thrice.

In vivo Angiogenesis Assay
In vivo angiogenesis was assayed by the Matrigel plug assay as described previously (24). These experiments were done under a protocol approved by the Institutional Animal Care and Use Committee at University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School. In brief, cold, low growth factor Matrigel (BD PharMingen, San Diego, CA, 500 µL per mouse) containing bFGF 400 ng/mL (R&D Systems), heparin 10 units/mL (Sigma, St. Louis, MO), and 108 plaque-forming units of adenoviral expression vector were injected into the flanks of C57BL/6 mice. After 14 days, the mice were euthanized by CO2 inhalation, and the plugs carefully removed en bloc with surrounding connective tissue. Tissue and plugs were fixed in cold acetone and frozen sections cut at 5 µm. Endogenous peroxidase activity was blocked with dilute H2O2. Sections were then blocked with 5% bovine serum albumin (BSA) for 15 minutes, washed with PBS, and then incubated with rat anti-mouse CD31 (PECAM) monoclonal antibody (BD PharMingen) in 1% BSA in PBS overnight. Sections were washed with cold PBS twice and incubated with biotinylated mouse anti-rat IgG1/2a (BD PharMingen) in 1% BSA/PBS. Color was then developed with streptavidin-peroxidase (VectaStain, ABC kit, Vector Laboratories, Burlingame, CA). Sections were counterstained with toluidine blue and vessel counts done as previously described (24, 29). In brief, vascular hotspots were located for each plug near the interface between the plug and surrounding stroma, and blood vessel density estimated as the number of CD31-positive cells per high-powered field. Two sections from each plug were made, at least five high-powered fields per section counted, and the mean ± SE determined for each experimental group. The experiment was repeated twice. Statistical differences were determined by one-way ANOVA using Prism v.4.0 (GraphPad Software, Inc., San Diego, CA), followed by Dunnett's multiple comparison test.

Genome-wide Gene Expression Profiling
We compared global gene expression in control HUVECs transduced with Ad.GFP with that of HUVECs transduced with Ad.rGax or Ad.hGax. Cells were transduced at a multiplicity of infection (MOI) of 100, incubated 24 hours in normal medium, then harvested for total RNA isolation as described above. RNA quality was verified by electrophoresis through formaldehyde-containing agarose gels before use for generating probes. Exogenous Gax expression was verified by Western blot (data not shown). Global gene expression was then compared in two separate experiments using the Affymetrix Human Genome U133A GeneChip array set and standard protocols supplied by the manufacturer, with technical assistance from the cDNA Microarray Core Facility of the Cancer Institute of New Jersey. The U133A chip contains probe sets for over 33,000 known genes, along with probes for housekeeping genes for normalization and genomic DNA for evaluation of hybridization quality. Results were analyzed using software provided by the manufacturer and then further analyzed with GeneMAPP (30) to identify signal pathway–dependent changes in gene expression.

Western Blots
Whole cell extracts from TNF-{alpha}-treated HUVECs were electrophoresed through 8% SDS-polyacrylamide gels and transferred to polyvinylidene diflouride membranes. The membranes were blocked with PBS plus 5% nonfat dry milk and 0.1% Tween 20 before being incubated with the appropriate dilution of primary antibody (mouse monoclonal anti-VCAM-1 and anti-ICAM-1 and rabbit polyclonal anti-E-selectin, Santa Cruz Biotechnology, Santa Cruz, CA) in blocking solution. Blots were washed with blocking solution and incubated with secondary antibody (goat anti-mouse IgG or goat anti-rabbit IgG; Pierce Biotechnology, Inc., Rockford, IL) and then washed again with blocking solution. Bands were visualized by chemiluminescence using the ECL-Plus reagent (Amersham, Piscataway, NJ).

Flow Cytometry
Cells were harvested after the relevant treatment and resuspended in PBS containing 0.1% sodium azide. Approximately 1 x 105 cells were incubated with FITC-conjugated primary antibody against human E-selectin, VCAM-1, or ICAM-1 (BD Biosciences, San Diego, CA) for 30 minutes on ice. Cells were pelleted and washed twice in PBS/azide before flow analysis on a Beckman-Coulter Cytomics FC500 flow cytometer (Fullerton, CA).

Electrophoretic Mobility Shift Assays
HUVECs were transduced overnight with Ad.GFP or Ad.rGax and then induced with 10 ng/mL TNF-{alpha} for 1 hour. Nuclear extracts were prepared with the NE-PER nuclear extraction reagent (Pierce Biotechnology) and incubated with a biotin end-labeled double-stranded oligonucleotide containing the NF-{kappa}B consensus sequence (5'-biotin-AGT TGA GGG GAC TTT CCC AGG C-3'; IDT DNA Technologies, Coralville, IA). The binding reactions, containing 6 to 8 µg of nuclear extract protein, buffer [10 mmol/L Tris (pH 7.5), 50 mmol/L KCl, 1 mmol/L DTT], 1 µg of poly(deoxyinosinic-deoxycytidylic acid), 5 µg BSA, and 20 fmol/L of biotin-labeled DNA, were incubated at room temperature for 20 minutes. Competition reactions were done by adding up to 200-fold excess unlabeled double-stranded NF-{kappa}B consensus oligonucleotide to the reaction mixture. Other controls included competition with random oligonucleotide (5'-TAG CAT ATG CTA-3') and an NF-{kappa}B site with a point mutation that abolishes DNA binding (5'-CAC AGT TGA GGC CAC TTT CCC AGG C-3'). Reactions were electrophoresed on a 6% acrylamide gel at 100 V for 1 hour in 0.5xTris-borate-EDTA buffer and then transferred to positively charged nylon membranes. Biotinylated oligonucleotides were detected with streptavidin-linked horseradish peroxidase and the Pierce LightShift kit (Pierce Biotechnology).


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Gax Expression Is Rapidly Down-regulated by Mitogens and Proangiogenic Factors in Endothelial Cells
We first wished to determine how Gax expression is regulated by growth factors and proangiogenic peptides in endothelial cells. HUVECs made quiescent by incubation for 24 hours in 0.1% FBS were stimulated with 10% FBS plus 5 ng/mL VEGF. Gax mRNA was rapidly down-regulated by 5-fold within 4 hours and slowly returned to basal over 24 to 48 hours (Fig. 1A and C). Conversely, when sparsely plated randomly cycling HUVECs were placed in medium containing 0.1% serum, Gax was up-regulated nearly 10-fold within 24 hours (Fig. 1B). Quiescent HUVECs were then stimulated with proangiogenic or proinflammatory factors, including bFGF, VEGF, and TNF-{alpha}. Gax was rapidly down-regulated with a similar time course (Fig. 2A). Similar results were observed in HMEC-1 cells (23), an immortalized human microvascular endothelial cell line (data not shown). Finally, conditioned medium from several breast cancer cell lines was used to stimulate quiescent HUVECs for 4 hours. The cell lines varied considerably in their ability to down-regulate Gax, but all of them down-regulated Gax expression at least 3-fold, and some by as much as 20-fold (Fig. 2B), suggesting that tumor-secreted proangiogenic factors also down-regulate Gax expression.



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  		Figure 2. Gax down-regulation by mitogens, proinflammatory factors, and tumor-secreted factors. A, Mitogens and proangiogenic factors cause rapid down-regulation of Gax expression in endothelial cells. Quiescent HUVECs were treated with either 10% FBS or 10 ng/mL of either VEGF165, TNF-{alpha}, or bFGF. At various time points, cells were harvested for extraction of total RNA, which was then subjected to quantitative real-time TaqMan RT-PCR with Gax- and GAPDH-specific primer/probe sets. (See Materials and Methods for sequences and details.) B, down-regulation of Gax expression in endothelial cells by conditioned medium from tumor cell lines. Quiescent HUVECs were treated with either low-serum medium, 10% FBS, or 10% conditioned medium from the indicated breast cancer cell lines. Cells were harvested 4 hours after stimulation, total RNA harvested, and real time quantitative RT-PCR done. All Gax mRNA levels were normalized to GAPDH expression, and units are arbitrary.

 
Gax Expression Inhibits Endothelial Cell Migration toward Proangiogenic Factors
Migration of endothelial cells through the basement membrane and into the surrounding stroma in response to proangiogenic stimuli is a critical step in tumor-induced angiogenesis. We therefore tested the ability of Gax to inhibit endothelial cell migration toward proangiogenic factors. HUVECs were transduced with Ad.rGax or Ad.hGax at varying MOI and incubated overnight. Viable cells (105 per well) were plated in six-well plates with inserts containing 8-µm polycarbonate filters and their migration toward angiogenic factor–containing media in the lower chamber measured. Ad.rGax strongly inhibited the migration of HUVECs toward serum, VEGF, bFGF, and TNF-{alpha} (Fig. 3), as did Ad.hGax (data not shown). Both homologues also inhibited migration of HMEC-1 cells toward bFGF and VEGF (data not shown).



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  		Figure 3. Gax inhibits HUVEC migration toward serum. HUVECs were transduced with varying MOIs of either Ad.GFP or Ad.rGax and their migration toward various growth factors and proangiogenic factors determined (see Materials and Methods). Gax inhibits HUVECs migrating toward (A) FBS; and (B) FBS, bFGF, VEGF165, and TNF-{alpha}. Results are expressed relative to control HUVECs not transduced with any virus. Results were analyzed by one-way ANOVA; *, P < 0.01. Similar results were obtained with Ad.hGax (data not shown).

 
Gax Expression Inhibits In vivo Angiogenesis
Matrigel containing proangiogenic factors, when implanted s.c. in mice, can stimulate the ingrowth of blood vessels into the Matrigel plug from the surrounding tissue, allowing in vivo tumor cell-free estimates of angiogenesis (24). Moreover, adenoviral vectors diluted in Matrigel implanted as s.c. plugs can serve as reservoirs to transduce endothelial cells invading the plug and drive expression of exogenous genes, producing effects on in vivo angiogenesis (31). We therefore used Matrigel plugs to test whether exogenously driven Gax expression can inhibit angiogenesis in vivo, using methodology previously described (24). Matrigel plugs containing bFGF and either Ad.GFP, Ad.hGax, or Ad.rGax (see Materials and Methods) were injected s.c. into C57BL/6 mice (n = 8 per experimental group). As a positive control for inhibition of angiogenesis in vivo by a viral vector, we used an additional adenoviral construct expressing a form of Akt (T308A, S473A, adenoviral construct designated Ad.DN.Akt) that functions in a dominant-negative fashion (24) and has previously been used in the Matrigel plug assay to show that inhibition of Akt signaling inhibits angiogenesis in vivo (24). As another control, to verify that adenovirus itself does not significantly alter in vivo angiogenesis as measured by this assay, plugs containing only bFGF were also examined. Adenoviral vectors expressing Gax expression were observed to inhibit the neovascularization of the plugs with a potency slightly less than what was observed for the Ad.DN-Akt construct (Fig. 4), and the Ad.DN.Akt construct inhibited neovascularization with a potency similar to what has previously been reported (24).



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  		Figure 4. Effect of Gax expression on angiogenesis in Matrigel plugs. Matrigel plugs (500 µL each) containing 400 ng/mL bFGF and the indicated viral constructs at 108 plaque-forming units per plug were implanted s.c. in the flanks of C57BL6 mice. Plugs were harvested after 14 days incubation for immunohistochemistry using CD31 antibodies and determination of CD31-postitive cells per high powered (400x) field (see Materials and Methods and Results for details). MG, Matrigel plug; ST, stroma surrounding the plug. Arrows, examples of CD31-positive blood vessels. A, Gax inhibits in vivo angiogenesis. Plugs with either no growth factor or bFGF plus Ad.GFP, Ad.dN.Akt, Ad.hGax, or Ad.rGax were implanted into the flanks of C57BL/6 mice (see Materials and Methods for details and concentrations). After 14 days, the mice were euthanized and the plugs harvested for immunohistochemistry with CD31. Immunohistochemistry using anti-Gax antibodies according to previously described methods (13) was done on a representative plug into which Ad.rGax had been introduced to show that the construct is transducing the cells within the plug (lower right hand corner). B, vessel counts. Columns, means; bars, SE. Statistical differences determined with one-way ANOVA; P < 0.0001 for the overall. The vessel counts were statistically significantly different from control (Ad.GFP group) for Ad.DN.Akt (P = 0.013), Ad.hGax (P = 0.008), and Ad.rGax (P = 0.028). C, gross photographs of selected plugs. Note the hemorrhage into one of the Ad.GFP plugs and the lack of vessels on the capsule of the Ad.Gax and Ad.dN.Akt plugs.

 
Gax Expression Down-regulates the Expression of NF-{kappa}B Target Genes
Next, in order to attempt to identify downstream targets and signaling pathways regulated by Gax expression, we determined differences in global gene expression between control HUVECs infected with Ad.GFP with HUVECs infected with Ad.rGax or Ad.hGax. Cells were infected at an MOI = 100, incubated 24 hours in normal media, then harvested for total RNA isolation. Global gene expression was compared in two separate experiments using the Affymetrix Human Genome U133A GeneChip array set (see Materials and Methods). We observed 127 probe sets corresponding to known genes showing greater than 2-fold up-regulation and 115 showing greater than 2-fold down-regulation. Differences in gene expression between controls and Gax-transduced cells ranged from up-regulation by approximately 30-fold to down-regulation by 239-fold. This pattern was similar in endothelial cells transduced by Ad.hGax, although the magnitude of changes in gene expression tended to be smaller (data not shown). We report here only probe sets that represent known genes that were either up- or down-regulated by at least 2.5-fold, with the addition of a few genes regulated <2.5-fold selected because they are either involved in angiogenesis, regulated by NF-{kappa}B, or both (Table 1).


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  		Table 1. Genes regulated by Gax expression

 
Consistent with the hypothesis that Gax inhibits endothelial cell activation, Gax strongly down-regulated several CXC chemokines (Table 1). Most strongly down-regulated of all was GRO-{alpha} (CXCL1), a CXC chemokine and a growth factor for melanoma that has also been implicated in promoting angiogenesis (32). Gax also down-regulated cell adhesion molecules known to be up-regulated in endothelial cells during activation and angiogenesis, including VCAM-1, ICAM-1, and E-selectin (33), all of whose down-regulation we have confirmed using real time quantitative RT-PCR, Western blot, and flow cytometry (Fig. 5). Moreover, Gax inhibited both the basal and TNF-{alpha}-induced up-regulation of ICAM-1, VCAM-1, and E-selectin proteins (Fig. 5C and D, and not shown). The pattern of down-regulation of these adhesion molecules, which are normally up-regulated during endothelial cell activation and angiogenesis, coupled with the down-regulation of CXC chemokines, suggested the inhibition of genes normally induced by TNF-{alpha}, which in turn suggested the possibility that Gax may inhibit NF-{kappa}B activity. Indeed, when our data was analyzed using GeneMAPP (30) to look for patterns of signal-dependent gene regulation, numerous NF-{kappa}B-dependent genes were identified (Table 1). Western blot analysis showed no difference between untransduced endothelial cells and cells transduced with Ad.GFP in either the TNF-{alpha}-induced expression of VCAM-1 or E-selectin (Fig. 5C) or the basal level of VCAM-1, ICAM-1, or E-selectin protein (not shown), and only slight differences by flow cytometry (Fig. 5D), suggesting that our result is not an artifact of our use of Ad.GFP as a control in the initial gene expression profiling experiment. Further supporting this conclusion is our observation by quantitative real time RT-PCR that (1) there was no difference between untransduced HUVECs and those transduced with Ad.GFP in the expression of E-selectin, ICAM-1, VCAM-1, Gro-{alpha}, VEGF-C, bFGF, p21CIP1/WAF1, and a variety of other genes identified in Table 1 as being regulated by Gax (data not shown); and (2) that the same result was obtained for Gro-{alpha}, E-selectin, and VCAM-1 using nonviral means of transducing the HUVECs in which no GFP-containing vectors were used (Fig. 5B).



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  		Figure 5. Effect of Gax expression on the level of E-selectin, VCAM-1, and ICAM-1. A, Gax down-regulates cell adhesion molecule mRNAs in HUVECs. HUVECs were transduced with Ad.GFP, Ad.hGax, or Ad.rGax, incubated for 24 hours in normal growth medium, then harvested for total RNA isolation. Total RNA was then subjected to quantitative real time RT-PCR using TaqMan primers and probes specific for each gene and the results normalized to GAPDH. A very strong down-regulation of E-selectin, VCAM-1, and ICAM-1 message level was observed. B, Gax down-regulates NF-{kappa}B-dependent genes using nonviral transduction. To rule out artifacts from GFP expression, HUVECs were transfected with pCGN-Gax or pCGN empty vector and then incubated overnight in growth medium. Cells were then harvested for total RNA, which was subjected to real time quantitative RT-PCR as described in Materials and Methods. Despite the lower transfection efficiency of liposomal-mediated methods, a strong down-regulation of NF-{kappa}B-dependent genes was observed compared with the empty vector. Units are arbitrary for (A) and (B, C). C, Gax down-regulates HUVEC expression of cell adhesion molecules. HUVECs were transduced with Ad.rGax or Ad.GFP and then incubated overnight, after which they were stimulated with 10 ng/mL TNF-{alpha} for 4 hours. Cells were harvested for total protein and subjected to Western blot with appropriate antibodies. Expression of Gax from the adenoviral vector was verified by Western blot with antibodies against Gax as previously described (13). Gax also down-regulated ICAM-1 (not shown). D, Gax down-regulates cell surface expression of ICAM-1, E-selectin, and VCAM-1. HUVECs transduced overnight with either Ad.GFP or Ad.rGax at an MOI = 100 were stimulated with TNF-{alpha} 10 ng/mL for 4 hours and then harvested for flow cytometry using appropriate antibodies (see Materials and Methods). Ad.rGax blocked the expression of VCAM-1, E-selectin, and ICAM-1.

 
In contrast, the genes up-regulated by Gax did not fall into any signal-dependent patterns as striking as the genes down-regulated by Gax (Table 1). However, there were still results that might suggest specific pathways up-regulated by Gax. First, there was a strong up-regulation of ALK3 (bone morphogenetic receptor 1a; 34). Although it is known that ALK1 activates endothelial cells through a SMAD1/5 pathway and ALK5 inhibits endothelial cell activation through a SMAD2/3 pathway (35), it is not known what role ALK3 plays in regulating endothelial cell phenotype. Second, we observed the up-regulation of three CDK inhibitors, p19INK4D, p57Kip2, and p21WAF1/CIP1 (10, 36, 37), consistent with a role in promoting cell cycle arrest and the quiescent phenotype. Finally, Frizzled-2 was strongly up-regulated. Little is known about the potential role of Frizzled receptors and Wnt signaling in regulating postnatal angiogenesis, although Frizzled-2 is expressed in endothelial cells (38) and there is evidence suggesting Wnt signaling inhibits endothelial cell proliferation (39).

Gax Expression Blocks NF-{kappa}B Binding to its Consensus DNA-Binding Sequence
Given that NF-{kappa}B activity has been implicated in the changes in phenotype and gene expression endothelial cells undergo during angiogenesis caused by VEGF, TNF-{alpha}, and other factors (16–22), we wished to confirm our findings from gene expression profiling that Gax inhibits NF-{kappa}B activity in endothelial cells. We therefore did electrophoretic mobility shift assays with a probe containing an NF-{kappa}B consensus sequence (40) utilizing nuclear extracts from HUVECs transduced with either Ad.rGax or the control adenoviral vector Ad.GFP. Gax expression in HUVECs markedly reduced specific binding to NF-{kappa}B consensus sequence by nuclear extracts compared with what was observed in controls (Fig. 6A), implying that Gax expression interferes with the binding of NF-{kappa}B to its consensus sequence. Unlabeled double-stranded NF-{kappa}B consensus oligonucleotide competed with labeled probe for binding (Fig. 6B), and random oligonucleotide and an NF-{kappa}B site with a point mutation that abolishes DNA binding (see Materials and Methods for sequences) failed to compete with the probe-specific band (data not shown).



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  		Figure 6. Gax expression inhibits NF-{kappa}B activity. A, Gax blocks NF-{kappa}B binding to its consensus sequence. HUVECs were infected with adenovirus containing GFP or rGax, incubated overnight in EGM-2, and then induced with 10 ng/mL TNF-{alpha} for 1 hour. Controls were not induced with TNF-{alpha}. Nuclear extracts were prepared and incubated with biotinylated oligonucleotides containing the consensus NF-{alpha}B binding site (see Materials and Methods). B, control electrophoretic mobility shift assay. Excess unlabeled wild-type NF-{kappa}B oligonucleotide competes with NF-{kappa}B probe. Random oligonucleotide and an NF-{kappa}B site with a point mutation that abolishes DNA binding (see Materials and Methods for sequences) failed to compete with the probe-specific band (data not shown). Moreover, Gax expression did not affect binding to an unrelated probe (Oct-1, data not shown). Arrows, NF-{kappa}B specific bands, and bands at the bottom of the gels represent unbound probe. NT, no treatment with TNF-{alpha}; NV, no virus; NE, no nuclear extract; NC, no unlabeled competitor; and WT, wild-type.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Interactions between tumors and their surrounding stroma, particularly the ability of tumors to induce angiogenesis, are critical to tumor progression and metastasis (41). At the endothelial cell level, the process of angiogenesis involves complex temporally coordinated changes in phenotype and global gene expression in response to alterations in the balance between pro- and antiangiogenic factors (2, 3). The stimuli for these changes are communicated from the surface of endothelial cells to the nucleus through multiple overlapping signaling pathways. The peptide factors and the receptors they bind to that activate these pathways have been the subject of intense study over the last decade, because the importance of aberrant endothelial cell activation and angiogenesis to the pathogenesis of not just cancer, but of other diverse human diseases, such as atherosclerosis, diabetic retinopathy, psoriasis, and others, has become more apparent (42). Because blocking aberrant angiogenesis has the potential to be an effective strategy to treat or prevent cancer and other angiogenesis-dependent diseases, understanding how downstream transcription factors integrate upstream signals from pro- and antiangiogenic factors to alter global gene expression and produce the activated, angiogenic phenotype, has become increasingly important.

Homeobox genes represent a class of transcription factors that, given their ubiquitous roles in controlling body plan formation during embryogenesis, organogenesis, cell proliferation and differentiation, and numerous other important cellular processes (5, 7), might be expected to be involved in either promoting or inhibiting the conversion of quiescent, unactivated endothelial cells to the activated, angiogenic phenotype. Indeed, several homeobox genes (HOXA9EC, HOXB3, HOXB5, HOXD3, HOXD10, and Hex) have already been implicated in this process (7, 43). We postulated that at least one additional homeobox gene, Gax, is also likely to play an important role in regulating endothelial cell angiogenesis. Consistent with its regulation in vascular smooth muscle cells, in endothelial cells, Gax is rapidly down-regulated by serum, proangiogenic, and proinflammatory factors (Figs. 1 and 2), and is able to inhibit endothelial cell migration in vitro (Fig. 3) and angiogenesis in vivo (Fig. 4). These observations led us to examine the mechanism by which Gax inhibits endothelial cell activation by examining global changes in gene expression due to Gax. In addition to observing that Gax up-regulates cyclin kinase inhibitors and down-regulates a number of proangiogenic factors, we also found that Gax inhibits the expression of NF-{kappa}B target genes (Table 1). Consistent with expression profiling data, Gax inhibits the binding of NF-{kappa}B to its consensus sequence (Fig. 6).

Several lines of evidence implicate NF-{kappa}B activity in regulating endothelial cell phenotype during inflammation and angiogenesis (16–19). For example, proangiogenic factors such as VEGF (33), TNF-{alpha} (44), and platelet-activating factor (17) can all activate NF-{kappa}B signaling and activity in endothelial cells. In addition, inhibition of NF-{kappa}B activity blocks tube formation in vitro on Matrigel (22), and pharmacologic inhibition of NF-{kappa}B activity suppresses retinal neovascularization in vivo in mice (45). Similarly, {alpha}5ß1-mediated adhesion to fibronectin also activates NF-{kappa}B signaling and is important for angiogenesis, and inhibition of NF-{kappa}B signaling inhibits bFGF-induced angiogenesis (16). One other potential mechanism by which NF-{kappa}B signaling may promote angiogenesis is through an autocrine effect, whereby activation of NF-{kappa}B induces expression of proangiogenic factors such as VEGF, as has been reported for platelet-activating factor–induced angiogenesis (17). Alternatively, the involvement of NF-{kappa}B in activating endothelial cell survival pathways is also likely to be important for sustaining angiogenesis (46).

Although NF-{kappa}B or I{kappa}B activity can regulate the expression of homeobox genes (47), there have been few reports of functional interactions between homeodomain-containing proteins and NF-{kappa}B or I{kappa}B proteins. The first such interaction reported was between I{kappa}B{alpha} and HOXB7, in which I{kappa}B{alpha} was reported to bind through its ankyrin repeats to the HOXB7 protein and thus potentiate HOXB7-dependent gene expression (48). In contrast, the POU factor Oct-1 can compete with NF-{kappa}B for binding to a specific binding site in the TNF-{alpha} promoter because its consensus sequence is close to the NF-{kappa}B consensus sequence (49). In addition, at least one interaction has been described in which a homeobox gene directly inhibits NF-{kappa}B-dependent gene expression, an interaction in which Cdx2 blocks activation of the cyclooxygenase-2 promoter by binding p65/RelA (50). It remains to be elucidated if Gax inhibits NF-{kappa}B-dependent gene expression by a similar mechanism. Regardless of the mechanism, however, this report represents to our knowledge the first description of a homeobox gene that not only inhibits the phenotypic changes that occur in endothelial cells in response to proangiogenic factors but also inhibits NF-{kappa}B–dependent gene expression in vascular endothelial cells while doing so. These properties suggest Gax as a potential important transcriptional inhibitor of endothelial cell activation and thus a potential target for the antiangiogenic therapy of cancer or other angiogenesis-dependent diseases. In addition, understanding the actions of Gax on downstream target genes, signals that activate or repress Gax expression, and how Gax regulates NF-{kappa}B activity in endothelial cells is likely to lead to a better understanding of the mechanisms of tumor-induced angiogenesis and the identification of new molecular targets for the antiangiogenic therapy of cancer.


   Acknowledgments
 
Grant support: New Jersey Commission on Cancer Research grant 0139CCRS1 and the U.S. Department of Defense grants DAMD17-02-1-0511 and DAMD17-03-1-0292.

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.

We thank Dr. Kenneth Walsh (Boston University) for anti-Gax antibody and for advice on performing the Matrigel plug assay, Dr. Daniel Medina for constructs and technical assistance with flow cytometry, and Dr. Arnold Rabson for his helpful advice on NF-{kappa}B (both of University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, New Brunswick, NJ).

Received 9/22/04. Revised 11/22/04. Accepted 12/ 7/04.


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