Dioxin Receptor Deficiency Impairs Angiogenesis by a Mechanism Involving VEGF-A Depletion in the Endothelium and Transforming Growth Factor-β Overexpression in the Stroma^*

Reagents and Cell Lines

Matrigel and FITC-labeled anti-CD102 were purchased from BD Biosciences and mouse recombinant VEGF-A₁₆₄ from Calbiochem. Heparin, endothelial cell growth factor supplement, recombinant TGFβ, neutralizing anti-VEGF-A and anti-β-actin antibodies, CoCl₂, and 2-methoxyestradiol (2-Me E2) were from Sigma-Aldrich. Antibodies against AhR and CD31 were purchased from Biomol and Abcam, respectively. A neutralizing anti-TGFβ antibody that recognizes all three TGFβ isoforms was obtained from R&D Systems (1D11 clone). Mouse melanoma B16F10 cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen), and human endothelial HMEC-1 cells used MCDB-131 medium (Invitrogen). Media were supplemented with 10% fetal bovine serum, 2 mm l-glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin.

The concentrations of VEGF-A used (100 ng/ml in aortic ring assays or 300 ng/ml in Matrigel plugs in vivo) were shown to efficiently modulate VEGF activity (35, 36). The anti-VEGF neutralizing antibody was used at 200 ng/ml based on the LD₅₀ provided by the manufacturer in human umbilical vein endothelial cells (50–150 ng/ml). Recombinant TGFβ was employed at 10 ng/ml taking into account our previous studies on cytokine activation in the ECM (37) and keratinocyte migration (38). The neutralizing anti-TGFβ antibody was used at 1 μg/ml considering its LD₅₀ (in-house determined to equal 60 ng/ml) (29) and its effect on keratinocyte migration (38).

Mice

AhR-null mice were produced by homologous recombination in embryonic stem cells as described (39). Mice were used at 10–12 weeks of age to obtain endothelial cells or aortic rings or at 30 weeks for in vivo Matrigel plug assays. The experiments involving animals were performed in compliance with the guidelines established by the Animal Care and Use Committee of the University of Extremadura.

Aortic Ring Assays and MAEC Isolation

Aortic Ring Assays and MAEC isolation were done as described previously (40) with some modifications. Briefly, the thoracic aorta was removed and sectioned in 1-mm aortic rings, which were embedded in Matrigel. After solidification, Dulbecco's modified Eagle's medium-F12 medium (Invitrogen) containing 20% fetal bovine serum, 200 units/ml penicillin, 200 μg/ml streptomycin, and 250 ng/ml endothelial cell growth supplement was added. The length of the sprouting vessels was measured at different times using light microscopy or 4,6-diamidino-2-phenylindole staining. MAECs were isolated at day 12 by solubilizing Matrigel-embedded aortic rings outgrowths in Matrisperse (BD Biosciences) followed by seeding in the same medium. MAEC cultures were assessed for purity by quantifying CD102-positive cells by flow cytometry as described previously (40). In some experiments, VEGF-A was added to the aortic rings at 100 ng/ml.

Induction of Hypoxia by Cobalt Chloride Treatment

To analyze the effects of hypoxia on VEGF expression, AhR^+/+ and AhR^−/− MAECs were cultured in complete medium and then treated with solvent (DMSO) or with 100 μm CoCl₂ for 16 or 24 h. RNA was purified and used to analyze VEGF mRNA levels as indicated below. This concentration of CoCl₂ has been shown to efficiently mimic hypoxia in cell culture (41). In these experiments, Vegf mRNA levels were normalized by β-actin because hypoxia can alter Gapdh expression (see primers used in Table 1). The contribution of hypoxia inducible factor-1α (HIF-1α) in maintaining VEGF levels was determined by adding 5 μm 2-Me E2 to CoCl₂-treated MAECs or to AhR small interfering RNA (siRNA)-transfected HMEC-1 cells. This concentration of 2-Me E2 was shown to induce degradation of HIF-1α in cultured cells (42, 43).

Tumor Xenografts

Aliquots of 5 × 10⁵ B16F10 cells (in 100 μl of phosphate-buffered saline) were injected subcutaneously into both flanks of five AhR^+/+ and AhR^−/− mice. Tumors were collected at 7 or 14 days after injection and their volume calculated as length × width² × 0.4. After fixation in 4% paraformaldehyde, tissues were sectioned at 5 μm and processed for hematoxylin and eosin staining or immunofluorescence.

In Vivo Matrigel Plug Assays and Hemoglobin Content

Aliquots consisting of 500 μl of ice-cold solution of 70% Matrigel, 30% Hanks' salt medium (Invitrogen), 30 units of heparin, and 300 ng of endothelial cell growth supplement were injected in duplicate into the flanks of at least four independent AhR^+/+ and AhR^−/− mice. One flank of each mouse was left as the control, and the other flank was used for the corresponding experimental treatments. In some experiments, VEGF-A₁₆₄ (300 ng/ml), TGFβ (10 ng/ml), a neutralizing anti-VEGF-A antibody (200 ng/ml), or a neutralizing anti-TGFβ antibody (1 μg/ml) were added to the Matrigel plugs. After 7 days, plugs were harvested, photographed, homogenized in 1 ml of ice-cold H₂O, and centrifuged at 10,000 × g for 6 min at 4 °C. The amounts of hemoglobin released to the supernatants were determined using Drabkin's reagent as indicated by the manufacturer (Sigma-Aldrich). Hemoglobin content is represented as mg/ml in 100 mg of Matrigel.

Isolation of Mouse Retina and Whole Mount Immunofluorescence

Retinas from five 3-day-old AhR^+/+ and AhR^−/− mice were isolated and processed as described previously (44). Briefly, eyes were collected and fixed overnight at 4 °C in Tris-buffered saline containing 4% paraformaldehyde. Retinas were dissected and permeabilized overnight at 4 °C in Tris-buffered saline containing 1% bovine serum albumin and 0.05% Triton X-100. After three washes in Tris-buffered saline, retinas were incubated overnight at 4 °C with isolectin B4-FITC (Sigma-Aldrich) in Tris-buffered saline. Flat-mounted retinas were analyzed by fluorescence microscopy. Measurements were taken near the optic nerve, at the center of the retina.

Hematoxylin/Eosin Staining and Tissue Immunofluorescence

Hematoxylin/eosin staining of tumor tissues was performed as indicated previously (33). For immunofluorescence, tissue sections were rehydrated, blocked, and incubated with CD31 antibody overnight at 4 °C. After washing in phosphate-buffered saline, sections were incubated with a TRITC-labeled secondary antibody (Sigma-Aldrich), mounted in Mowiol, and photographed under fluorescence microscopy.

In Vitro Tube Formation and Co-culture Experiments

Matrigel (70-μl aliquots) was gelled at 37 °C for 1 h. Then, 10⁵ MAECs were plated in OptiMEM (Invitrogen), and the formation of capillary-like structures was photographed 24 h later. For endothelial-mesenchymal co-culture experiments, AhR^+/+ and AhR^−/− immortalized T-FGM fibroblasts (33) were treated with mitomycin (Sigma-Aldrich) to inhibit cell proliferation. A sterile plastic ring was placed in a 35-mm tissue culture plate, and its interior volume was filled with Matrigel. After gelling, a monolayer of T-FGM AhR^+/+ or T-FGM AhR^−/− fibroblasts was seeded in the culture area outside the ring using OptiMEM. After 48 h, 10⁵ HMEC-1 cells were plated on the Matrigel plug located inside the ring. Once the endothelial cells adhered to the Matrigel, the ring was removed, and 16 h later, the formation of capillary-like structures was photographed. Experiments were also performed using T-FGM conditioned media rather than the cells themselves. T-FGM AhR^+/+ and T-FGM AhR^−/− fibroblasts growing at 30% confluence were cultured for 48 h in OptiMEM. Conditioned OptiMEM was then collected, processed as indicated previously (29), and used to analyze the formation of capillary-like structures as described above.

Measurement of Total Tube Length (TTL) and Calculation of Branching Points

TTL and branching points were measured in aortic ring explants and in Matrigel plugs by blinded analyses as described in Fig. 2A. Raw images were analyzed using ImageJ software to identify tubular structures. These images were subsequently employed to calculate TTL as the total length of the individual tubes present and branching as the number of bifurcations along the tubes (Fig. 2A, arrows).

Cell Viability and Cell Adhesion

After seeding for 16 h, 10⁵ HMEC-1 cells were washed, fixed, and stained with crystal violet. Following extensive washing with water, cultures were photographed and the number of viable cells counted. For adhesion experiments, aliquots of 10⁵ HMEC-1 cells were plated on gelled Matrigel plugs. After incubation at 37 °C for 1 h, cells were fixed, stained with crystal violet, and analyzed as indicated above.

Invasion in Matrigel Chambers and Wound Closure in Vitro

Invasion assays were done essentially as described (33). Briefly, 5 × 10⁴ HMEC-1 cells were plated in the upper chamber of a Matrigel invasion Transwell (BD Biosciences) in either OptiMEM or fibroblast-conditioned medium. Dulbecco's modified Eagle's medium-F12 medium containing 10 ng/ml insulin-like growth factor-1 (Sigma-Aldrich) was added to the lower chamber as a chemoattractant. After 24 h, Transwells were washed with phosphate-buffered saline, fixed at 4 °C in 70% ethanol, treated for 15 min with RNase (10 ng/ml), and stained with propidium iodide. Cell invasion into Matrigel was analyzed in 5-μm steps using a Zeiss LSM 510 confocal microscope. Wound closure experiments were performed using HMEC-1 in the absence or presence of AhR siRNA essentially as described (25, 33).

RNA Interference (RNAi) and Western Immunoblotting

siRNA for AhR or unspecific scramble RNA (Dharmacon) was transiently transfected in HMEC-1 cells by electroporation using a MicroPorator MP-100 (Digital-Bio). siRNA or scramble RNA were transfected at 100 nm using three pulses of 10 ms with the voltage set at 1400 V. HMEC-1 cells were grown for 60 h and then trypsinized and used for in vitro Matrigel tube formation. A fraction of the transfected cells was used to analyze AhR expression by Western immunoblotting as described (45).

Real-time PCR and ELISAs

Quantitative real-time RT-PCR and data analyses for the expression of angiogenesis-related genes were done as described earlier (45). Oligonucleotide sequences used are indicated in Table 1. The amounts of active VEGF-A, TGFβ-1, and TGFβ-2 secreted by MAECs or T-FGM fibroblasts were determined using ELISA kits from Bender MedSystems following the manufacturer's instructions. To activate latent TGFβ, conditioned medium was treated for 10 min at room temperature with 165 mm HCl and then neutralized with NaOH.

Image Processing and Statistical Analyses

Light and fluorescence microscopy measurements of aortic rings and branching of capillary-like structures in Matrigel plugs were quantified in at least five random fields using ImageJ software. All determinations were done in triplicate in at least two independent experiments. Data are shown as mean ± S.D. Statistical comparison between experimental conditions was done using GraphPad Prism 4.0 software. A comparison between conditions was done using the unpaired Student's t test, and multi-comparison was done using one-way analysis of variance followed by Dunn's post test.

Loss of AhR Expression Impairs Tumor Growth and Compromises Angiogenesis in Vivo

In previous work studying the potential of AhR-null transformed fibroblasts to induce tumors in mice, we suggested that lack of AhR expression could compromise tumor development by reducing cell migration and/or angiogenesis (33). These results prompted us to analyze whether AhR modulates angiogenesis and how its expression in the endothelial and stromal compartments contributes to the process. We first performed xenografts of B16F10 mouse melanoma cells in the dorsal area of AhR^+/+ and AhR^−/− mice (Fig. 1A). Tumors produced by B16F10 cells in AhR-null mice had a significantly reduced volume as compared with those isolated from AhR^+/+ animals at either 7 or 14 days after injection. In agreement to our hypothesis, this lower ability of AhR^−/− mice to support tumorigenesis could be related to impaired angiogenesis because quantification of vessel area by both hematoxylin and eosin staining and CD31 labeling of endothelial cells revealed that AhR^−/− tumors had significantly lower vessel numbers than wild-type tumors (Fig. 1B). Please note that hematoxylin and eosin images appear dark because of the high expression of melanin by B16F10 melanoma cells. To further support these observations, we next used an additional angiogenesis model based on the recruitment of blood vessels to Matrigel plugs inserted under the dorsal skin of AhR^+/+ and AhR^−/− mice. Consistently, the vascular system of AhR^−/− mice had a lower efficiency to produce new vessels able to colonize the Matrigel plug (Fig. 1C). Thus, loss of AhR expression appears to compromise angiogenesis in vivo.

Endothelial Cells Lacking AhR Have Altered Tube Formation and Branching

Two important factors that modulate efficient angiogenesis are the ability of the endothelial cells to branch and to form capillary tubes (4, 9, 46). We investigated whether deficient angiogenesis in AhR^−/− mice is related to altered branching and/or to the formation of tubular structures. Quantification of the total tube length (47) and of the number of branch points was performed in aortic rings and Matrigel plugs as indicated under “Experimental Procedures” and in the scheme shown in Fig. 2A. Culture of aortic rings (ar) from AhR^+/+ and AhR^−/− mice on Matrigel revealed that AhR-null MAECs were significantly impaired in their ability to form tubes as determined by measuring sprouting from the aorta (Fig. 2B). However, reduced TTL in AhR^−/− aortas was not due to a lower migration potential of AhR^−/− MAECs, as they invaded Matrigel to a similar extend as AhR^+/+ MAECs (Fig. 2B). Staining the Matrigel plug with 4,6-diamidino-2-phenylindole showed that whereas AhR^+/+ MAECs could orientate and align to form tubes, AhR^−/− MAECs emigrated from the aortic ring but remained mostly dispersed and formed fewer tubes (Fig. 2C). We then decided to analyze branching in AhR^−/− MAECs. MAECs were isolated from expanding aortic rings and plated in Matrigel (Fig. 3A). AhR-null cells not only formed a smaller amount of tubes but also exhibited a significantly reduced efficiency to branch as compared with AhR^+/+ MAECs. To confirm these results, vascular branching was also measured in retinas isolated from 3-day-old newborn mice. To avoid variations due to heterogeneity, measurements were taken near to the optic nerve, at the center of the retina. Fig. 3B shows that isolectin B4-FITC-labeled AhR^−/− retinas had fewer branching points per field than retinas from AhR^+/+ mice. Altogether, these experiments suggest that AhR expression is relevant to promote and to maintain angiogenesis, probably through the modulation of tube formation and branching of endothelial cells. The causal role of AhR in tube formation and branching was demonstrated by down-modulating AhR expression with RNAi. Human endothelial cells (HMEC-1) constitutively expressed AhR, and transfection of siRNA against this receptor significantly decreased its protein levels with respect to unspecific scramble-transfected cells (Fig. 3C). AhR was shown to be responsible for endothelial tube formation and branching, because siRNA-transfected HMEC-1 cells mimicked the phenotype observed in AhR^−/− MAECs (Fig. 3D). Reduced branching and impaired tube formation in the absence of AhR could be due to a decrease in cell viability and/or migration potential. However, down-modulation of AhR expression by siRNA did not significantly reduced viability or proliferation of HMEC-1 cells within a 72-h period (Fig. 3E). Further, wound healing in culture revealed that AhR siRNA did not affect the migration potential of HMEC-1 (Fig. 3F).

VEGF-A Expression Is Down-modulated in AhR^−/− MAECs: Role of Hypoxia and HIF-1α

VEGF is a major growth factor involved in angiogenesis and in vascular homeostasis (7, 9, 10). Because loss of AhR expression markedly altered angiogenesis in vivo and tubulogenesis and branching both in vivo and in culture, we decided to analyze whether the AhR^−/− phenotype was related to altered VEGF expression and/or activity. Real-time RT-PCR was used to measure mRNA expression of the angiogenesis-related genes Hif-1α, Vegf-A, Vegf-B, Plgf, Vegfr-1, and Vegfr-2. AhR^−/− MAECs had a significant decrease in Vegf-A and, to a lesser extent, Vegf-B expression. Interestingly, the expression of the VEGF-A-target gene Hif-1α was also reduced in AhR^−/− MAECs, suggesting that down-modulation of Vegf-A was functionally relevant (Fig. 4A). VEGF-A has several splice variants: VEGF₁₈₈, which is slightly active and tightly bound to the extracellular matrix, and VEGF₁₂₀ and VEGF₁₆₄, which are diffusible forms able to induce angiogenesis in vivo (48, 49). Remarkably, AhR^−/− MAECs had a significant down-modulation of Vegf₁₆₄ and Vegf₁₂₀ mRNAs as compared with AhR^+/+ MAECs, with Vegf₁₆₄ being the predominant isoform in these primary cells (Fig. 4B). Furthermore, the level of VEGF-A activity secreted to the medium (e.g. VEGF₁₆₄ and VEGF₁₂₀) was markedly reduced in AhR^−/− MAECs as compared with wild-type MAECs (Fig. 4C), suggesting that lower levels of VEGF-A could be relevant to the defective angiogenesis observed in AhR-null mice. In addition, the high level of Vegf₁₈₈ produced by AhR^−/− MAECs did not seem to compensate for the impairment in angiogenesis, further suggesting that Vegf₁₈₈ is not functionally relevant and that the reduction in Vegf₁₆₄ and Vegf₁₂₀ activities was mainly responsible for the lower angiogenic potential of MAECs.

The reduced expression of Hif-1α in AhR^−/− MAECs suggested that their deficiency in producing active VEGF and inducing angiogenesis could be due, at least in part, to an impaired response to hypoxia. To address this possibility, we treated AhR^+/+ and AhR^−/− MAECs with CoCl₂ to mimic hypoxia and then determined Vegf mRNA expression. Hypoxia readily increased Vegf expression with time in AhR^+/+ MAECs, whereas no significant induction was observed in AhR^−/− MAECs (Fig. 4D). Moreover, HIF-1α was involved in this mechanism, because treatment with 2-Me E2, which induces HIF-1α protein degradation, significantly blocked Vegf induction by CoCl₂ in AhR^+/+ but not in AhR−/− MAECs (Fig. 4D). The causal role of AhR in HIF-1α-mediated control of Vegf expression was further analyzed using HMEC-1 cells, which were found to respond to AhR down-modulation by RNAi (see Fig. 3). Transfection of HMEC-1 cells with AhR siRNA reduced the mRNA expression of both Hif-1α and Vegf-A (Fig. 5A). Accordingly, treatment with 2-Me E2 decreased Vegf levels in mock-transfected (scramble) but not in AhR siRNA-transfected HMEC-1 cells (Fig. 5B). Thus, reduced angiogenesis in AhR^−/− endothelial cells seems to be the result of lower VEGF-A production due to decreased HIF-1α expression.

Based on these results, we next used recombinant VEGF protein (purified VEGF₁₆₄, R&D Systems) and an inhibitory anti-VEGF antibody (neutralizes VEGF₁₆₄ and VEGF₁₂₀; Sigma-Aldrich) in an attempt to modulate angiogenesis in culture and in vivo. The addition of anti-VEGF antibody to AhR^+/+ aortic rings decreased the TTL to values similar to those found in AhR^−/− aortic rings, whereas treatment of AhR^−/− rings with recombinant VEGF protein did the opposite and rescued TTL levels to AhR^+/+ values (Fig. 6A). In vivo, the addition of anti-VEGF antibody to Matrigel plugs implanted in AhR^+/+ mice markedly reduced angiogenesis, whereas recombinant VEGF protein induced a modest, although very reproducible increase in blood vessels recruitment in plugs injected in AhR^−/− mice (Fig. 6B).

Increased Secretion of TGFβ Activity by Stromal Fibroblasts Also Contributes to Defective Angiogenesis in AhR^−/− Mice

TGFβ is a cytokine with a relevant role in angiogenesis that cooperates functionally with VEGF in endothelial cell function (16, 17, 22). Because we had found previously that stromal fibroblasts from AhR^−/− mice secrete increased levels of active TGFβ (25, 29), here we established co-cultures of Matrigel-embedded HMEC-1 cells and fibroblasts isolated from AhR^+/+ or AhR^−/− mice (Fig. 7A). HMEC-1 cells co-cultured with AhR^−/− fibroblasts lost their ability to form tubes as compared with the same endothelial cells co-cultured with AhR^+/+ fibroblasts (Fig. 7B). Moreover, such an effect on the endothelial cells was produced by a soluble and secreted molecule, because conditioned medium (CM) from AhR^−/− fibroblasts mimicked the effects induced by the fibroblasts themselves (Fig. 7C).

AhR^−/− fibroblasts expressed slightly increased levels of Tgfβ-1 and ∼2-fold more Tgfβ-2 mRNA than wild-type cells as determined by real-time RT-PCR (Fig. 8A, left). Nevertheless, TGFβ has to be secreted and activated in the extracellular matrix (15, 50) to exert its effects, and because AhR-null fibroblasts produce elevated levels of TGFβ activity (25, 29, 37, 51), we determined the amounts of active and total TGFβ-1 and TGFβ-2 released by AhR^+/+ and AhR^−/− fibroblasts by ELISA. We found that the total amount of TGFβ-1 secreted to the medium was markedly higher than that of TGFβ-2 in both genotypes (Fig. 8A, center and right). Importantly, AhR^−/− fibroblasts released higher levels of both active and total TGFβ-1 than AhR^+/+ cells, although no significant differences were observed with respect to active TGFβ-2 (Fig. 8A, center and right). Thus, increased production of TGFβ-1/2 by estromal fibroblasts could contribute to the inhibition of angiogenesis. Consistent with this hypothesis, the addition of recombinant TGFβ to medium conditioned by AhR^+/+ fibroblasts inhibited tube formation by HMEC-1 cells, whereas a neutralizing anti-TGFβ antibody added to medium conditioned by AhR^−/− fibroblasts increased tube formation (Fig. 8B). These detrimental effects on angiogenesis were not due to an inhibitory activity of the AhR^−/− conditioned medium or the TGFβ recombinant protein on HMEC-1 viability (Fig. 8C) or adhesion to Matrigel (Fig. 8D).

Because angiogenesis requires endothelial cell invasion of the surrounding tissue, we next used HMEC-1 cells as a model to analyze their invasion potential in the presence of medium conditioned by AhR^−/− fibroblasts and to determine whether TGFβ would contribute to such effect. HMEC-1 cells were plated in Matrigel-coated Transwells, and their invasive ability was determined by confocal microscopy. We found that the addition of medium conditioned by AhR^−/− fibroblasts to the upper chamber significantly inhibited HMEC-1 invasion as compared with endothelial cells treated with medium from wild-type fibroblasts (Fig. 9A). Furthermore, the addition of recombinant TGFβ to the medium conditioned by AhR^+/+ fibroblasts decreased HMEC-1 invasion, whereas a neutralizing anti-TGFβ antibody did the opposite, increasing HMEC-1 invasion in the presence of medium conditioned by AhR^−/− fibroblasts (Fig. 9A). Consistently, recombinant TGFβ was able to decrease blood vessel recruitment to Matrigel plugs injected in AhR^+/+ mice, whereas a neutralizing anti-TGFβ antibody enhanced vessel recruitment to Matrigel inserts in AhR^−/− mice (Fig. 9B). Importantly, VEGF and TGFβ acted together in AhR-dependent angiogenesis, because co-treatment of AhR^+/+ mice with anti-VEGF antibody plus TGFβ decreased blood vessel recruitment to Matrigel plugs, whereas the addition of VEGF plus anti-TGFβ antibody increased angiogenesis in Matrigel plugs implanted in AhR^−/− mice (Fig. 9B). This possible interaction between VEGF and TGFβ activities in endothelial cells is also supported by the observation that treatment of AhR^−/− MAECs with exogenous TGFβ further reduced their basal low level of VEGF-A secretion (Fig. 9C). Altogether, these data strongly suggest that TGFβ activity secreted to the extracellular medium by stromal fibroblasts contributes significantly to angiogenesis and that overactivation of TGFβ in the absence of AhR is a relevant factor in explaining their angiogenesis deficiency. In addition, the fact that an increase in VEGF cooperated with decreased TGFβ activity in improving angiogenesis in AhR^−/− mice (or the opposite in wild-type mice) further supports a functional interaction between endothelial and stromal cells in vessel recruitment and underlines AhR as a regulator of the process.