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* Department of Genetics and Pathology, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden;
# Department of Immunology, ImClone Systems Inc., New York, USA; and
Molecular/Cancer Biology Laboratory, Biomedicum Helsinki, University of Helsinki, Helsinki, Finland
1 Correspondence: Department of Genetics and Pathology, Vascular Biology Unit, Rudbeck Laboratory, Uppsala University, S-751 85 Uppsala, Sweden. E-mail: Stefan.Wennstrom{at}genpat.uu.se
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: embryoid body • endothelial cell • hypoxia • VEGFR-3
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. In the mouse yolk sac, the (hem)angioblasts form foci, called blood islands, composed of immature hematopoietic cells surrounded by primitive endothelial cells on embryonal day 6.5 (E6.5). Subsequent fusion of blood islands and differentiation result in formation of a primitive vascular plexus during mouse E8.0–8.5. In the embryo proper, migratory endothelial precursors aggregate into solid endothelial strands, which develop further to form the dorsal aorta, cardinal veins, and other vascular structures (2)
. In a second process, denoted angiogenesis, a functional network is formed through sprouting and pruning of new blood vessels from preexisting capillaries (3)
and by formation and insertion of tissue folds and columns of interstitial tissue into vessel lumen (intussuception) (4
).
Formation of the vascular system is regulated by various growth factors. Among the most important components are members belonging to the vascular endothelial growth factor (VEGF) family. In addition to VEGF-A, the founding member, the VEGF family includes four other mammalian ligands, VEGF-B, -C, -D and placental growth factor (PlGF). The VEGF ligands bind in a specific manner to three receptor tyrosine kinases, VEGFR-1 (also known as Flt-1), VEGFR-2 (Flk-1/KDR), and VEGFR-3 (Flt-4), which are expressed preferentially on blood and lymphatic endothelial cells (5
, 6)
. The importance of VEGF activity for proper vascular development has been thoroughly underscored in several gene deletion studies. For instance, heterozygous Vegf-a+/– mice exhibited embryonic lethality due to deficient intra- and extraembryonal vessel formation, indicating a dose-dependent regulation of embryonic vascularization by this factor (7
, 8)
. Furthermore, targeted disruption of the three Vegf receptor genes each resulted in various degrees of vascular abnormalities (9
10
11
12)
. In addition to VEGFs, several other factors play important roles in vascular development. Vital components for vessel remodeling and maturation are the angiopoietins and Tie receptors (13)
, ephrins and Eph receptors (14)
, and platelet-derived growth factors and their receptors (15)
.
The mammalian embryo develops in a relatively hypoxic environment; before implantation it relies on simple diffusion of oxygen, glucose, and nutrients. After onset of vascularization, the circulatory system still has to continuously evolve in order to satisfy the increasing oxygen and nutrient demand from rapidly expanding embryonic tissues. The developing embryo therefore experiences conditions in or close to the hypoxic range for most of the time prior to parturition (16)
. The most striking cellular response to hypoxia is transcriptional activation of a number of genes. The protein products of these genes, of which VEGF-A is one of the best characterized (17)
, regulate metabolic adaptation and cell survival as well as improve oxygenation, via erythropoiesis, and neovascularization (18)
. Although hypoxia-induced transcription has recently been elucidated at the molecular level (19)
, our understanding of how oxygen tension affects biological processes, such as embryonal development, is only beginning to emerge.
To study the effect of hypoxia on early vascular development we used differentiating mouse embryonal stem (ES) cells as a model. In the absence of leukemia inhibitory factor (LIF), ES cells aggregate and differentiate spontaneously into 3-dimensional structures, denoted embryoid bodies (EBs). Several aspects of early embryogenesis, including vasculogenesis and angiogenesis, can be recapitulated in EBs in a manner kinetically and morphologically resembling that in vivo (20
21
22
23
24
25
26
27)
.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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was routine-cultured on mitomycin C-arrested mouse embryonal fibroblasts in ES medium (DMEM-Glutamax supplemented with 15% fetal bovine serum, 25 mM HEPES pH 7.4, 1.2 mM sodium pyruvate, 0.12% monothiolglycerol, and 1000 U/mL LIF) and passaged every 48 h. All medium components were from Invitrogen (San Diego, CA, USA). At day 0, ES cells were trypsinated, resuspended in ES medium without LIF, and cultured in drops hanging from the lid of a nonadherent culture dish (1200 cells/drop) placed over a cell culture dish filled with phosphate-buffered saline. After 4 days, drops were collected and EBs were seeded out individually on 8-well chamber glass slides or in groups on 10 cm dishes (Becton Dickinson, Franklin Lakes, NJ, USA). EBs were maintained under normal growth conditions or incubated with VEGF-A165 (30 ng/mL; PeproTech, Rocky Hill, NJ, USA) or neutralizing VEGF receptor antibodies for the periods indicated. EBs subjected to hypoxia were placed in a humidified chamber and flushed with a 95% nitrogen/5% carbon dioxide gas mixture until 1% O2 was reached, as measured by a Pac III instrument (Dräger, Pittsburgh, PA, USA) placed inside the chamber. The chamber was sealed, incubated at 37°C, and the oxygen level continuously monitored by the Pac III instrument.
Neutralizing antibodies and proteins
The following neutralizing VEGF receptor antibodies were used: anti-VEGFR-1 (MF-1), anti-VEGFR-2 (DC101), and anti-VEGFR-3 (31C1). In addition, a VEGFR-3 chimeric protein, consisting of the extracellular ligand binding portion of VEGFR-3 joined to the Fc domain of immunoglobulin 
-chain, was used to sequester VEGFR-3 specific ligands (29)
. For neutralization, 30 µg/mL of the MF-1 and DC-101, 15 µg/mL of the 31C1, and 1 µg/mL of the VEGFR-3-Ig antibody were added every 48 h.
Immunohistochemistry
EBs were fixed in Zink buffer (37 mM ZnCl2, 23 mM ZnAc, 3.2 mM CaAc, and 0.2% Triton X-100), then subjected to chromogen staining. Endogenous peroxidase activity was quenched with 3% H2O2 in methanol and EBs were incubated with antibodies against CD31 (Becton Dickinson or Santa Cruz Biotechnology, Santa Cruz, CA, USA). After incubation with biotinylated secondary antibodies and streptavidin-conjugated horseradish peroxidase (HRP; both Vector Laboratories, Burlingame, CA, USA), immune reactivity was visualized using the AEC peroxidase substrate kit (Vector Laboratories). EBs were mounted with Ultramount aqueous mounting medium (DAKO, Copenhagen, Denmark) and analyzed by an Eclipse E1000 microscope (Nikon). Quantification of CD31-stained vascular areas was performed using the Easy Image Analysis 2000 software (Tekno Optik, Huddinge, Sweden). For fluorescent staining, EBs were fixed in Zink buffer or in cold 1:1 acetone/ethanol solution (for VEGFR-3 staining) and incubated with antibodies against CD31 (Becton Dickinson), Prox-1, VE-cadherin (R&D Systems, Abingdon, Oxon, UK), or VEGFR-3 (AFL4) (30)
. To detect VEGFR-3, a biotinylated secondary antibody (Vector Laboratories) and streptavidin-conjugated Alexa 488 (Molecular Probes, Eugene, OR, USA) were used. Otherwise, EBs were incubated with Alexa 488 and 568 secondary antibodies (Molecular Probes) before being mounted in Fluoromount (Southern Biotechnology, Birmingham, AL, USA) and analyzed by an Eclipse E1000 microscope (Nikon).
Magnetic cell sorting (MACS)
150–200 EBs were dissociated at 37°C using collagenase (2.5 mg/mL; Sigma) and cell clumps were dispersed by passing them through a 0.8 mm needle. To ensure single cell suspension, cells were passed through a 30 µm filter (Becton Dickinson). The cell suspension was incubated with rat CD31-FITC- and rat VEGFR-2-PE-conjugated antibodies (Becton Dickinson) before being incubated with magnetic goat anti-rat IgG microbeads (Miltenyi Biotec, Auburn, CA, USA). CD31- and VEGFR-2-positive cells were purified using MS separation columns (Miltenyi Biotec) according to the manufacturer’s instructions, except that an extra column step was included to increase the purity of isolated endothelial cells.
Immunoblotting
EBs were lysed in Nonidet P-40 (NP-40) lysis buffer (50 mM HEPES pH 7.5, 100 mM NaCl, 1 mM EGTA, 1 mM DTT, 1 mM PMSF, 5 µg/mL aprotinin, 5 µg/mL leupeptin, 100 µM Na3VO4, and 1% NP-40) and lysates were subjected to SDS-PAGE, followed by transfer to Hybond-C extra membranes (Amersham Biosciences, Uppsala, Sweden). Membranes were incubated with antibodies against CD31 (Santa Cruz Biotechnology), VEGFR-1, VEGFR-2 (Santa Cruz Biotechnology), or VEGFR-3 (AFL4). Antibodies against ß-actin (Santa Cruz Biotechnology) were used to verify comparable loading of cell lysates. Membranes were incubated with HRP-conjugated secondary antibodies (Amersham Biosciences) and immune reactivity was visualized using the enhanced chemiluminescence (ECL) detection system (Amersham Biosciences).
Real-time and reverse transcriptase polymerase chain reaction (PCR)
Total RNA, prepared from EBs using the RNeasy mini kit (Qiagen, Hilden, Germany), was treated with DNase I (Amersham Biosciences) and used for first-strand cDNA synthesis using oligo dT primers and the Advantage RT-for-PCR-Kit (Clontech, Palo Alto, CA, USA). PCR primers, according to Table 1
, were designed using the Primer Express software (Applied Biosystems, Foster City, CA, USA) and BLAST searches were performed for each primer to avoid sequence homology with other genes. All primers were from Invitrogen. The cDNA was mixed with primers and SYBR Green PCR master mix (Applied Biosystems), and amplified by PCR using an ABI Prism 7700 instrument (Applied Biosystems). PCR conditions were 95°C 10 min – (95°C 15 s–60°C 1 min) x 45. The calculated threshold cycle (CT) value for each transcript was normalized against the corresponding ß-actin CT value. Identical conditions were used in reverse transcriptase PCR, except for different cycle numbers.
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Statistical analysis
Immunostainings and immunoblots show representative results from experiments repeated at least five times. Quantification of vascular area was performed on 10 EBs per condition and is given as mean values ±SD. Real-time PCR data of transcript levels show representative results and are given as the mean induction level of triplicates ± SD compared with normoxic control (set to 1.0). The expression profile for each gene was analyzed using at least three different RNA preparations.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The vasculature was visualized by immunohistochemical staining for the endothelial cell marker CD31/platelet endothelial cell adhesion molecule-1. EBs cultured for 8 days in normoxia displayed immature clusters of CD31-positive endothelial cells (Fig. 2
A, B). In contrast, EBs cultured in hypoxia between day 6 and 8 had developed a plexus of capillary-like structures. Administration of exogenous VEGF-A promoted formation of a peripheral and relatively unorganized network.
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Hypoxia treatment of EBs between day 8 and 12 resulted in an expansion of the vascular network characterized by a dramatic stimulation of cell fusion and formation of a dense mesh (Fig. 2A, B
). In contrast, in normoxic EBs treated with VEGF-A during the same period, the development appeared to be halted and CD31-positive cells failed to organize into vessel-like structures. Quantification of CD31-stained areas revealed a clear difference between normoxic, hypoxic, and VEGF-A-treated EBs (Fig. 2C
). If EBs were placed in hypoxia from day 6 to day 10 or 12, the resulting vascular network was not as extensive as the one formed between day 8 and 12 (data not shown). Thus, the unique influence of hypoxia on vascularization, which was confined to a discrete developmental stage, was morphologically different from that induced by VEGF-A.
Hypoxia alters expression of vascular factors and endothelial cell markers
To study expression of VEGF signaling components and endothelial cell markers during vascular network formation, we extracted RNA from EBs subjected to normoxia, hypoxia, or VEGF-A treatment between day 8 and 12. The RNA was used to analyze steady-state transcript levels by quantitative real-time PCR. As seen in Fig. 3
A, hypoxia markedly increased VEGF-A and VEGFR-1 transcript levels. In contrast, EBs treated with VEGF-A exhibited a clear reduction in expression from the Vegf-a and Vegfr-1 genes. Expression of placental growth factor (PlGF), a VEGFR-1-specific ligand, remained unchanged, whereas both hypoxia and VEGF-A reduced the number of VEGFR-2 transcripts. VEGFR-3 and one if its ligand, VEGF-D, were significantly up-regulated in hypoxic EBs, while VEGF-C expression was not induced at this stage. Analysis of endothelial cell markers CD31 and vascular endothelial-cadherin (VE-cadherin) revealed a 2-fold induction in hypoxic EBs. No effect on these two endothelial cell markers was observed in VEGF-A-treated EBs.
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We next analyzed protein levels for some of the quantified transcripts by immunoblotting of total EB lysates. Overall, changes in transcript levels correlated well with changes in protein levels in that VEGFR-3 and CD31 protein levels were strongly up-regulated by hypoxia whereas VEGFR-2 levels were markedly reduced in both hypoxic and VEGF-A-treated EBs (Fig. 3B
). An induction, although weak, was seen for the VEGFR-1 protein. In conclusion, hypoxia-induced vascular development is accompanied by up-regulation of several VEGF genes and endothelial cell markers.
Hypoxia increases VEGFR-3 expression on vascular endothelial cells
To analyze gene expression specifically in endothelial cells, normoxic and hypoxic EBs were collagenase-treated and endothelial cells isolated by MACS using CD31 and VEGFR-2 antibodies. RNA was prepared and transcript levels analyzed by quantitative real-time PCR. As seen in Fig. 4
A, the differential effect of hypoxia on VEGFR-1, -2, and -3 expression observed in EBs was clearly manifested in the endothelial cell pool. In contrast, hypoxia had no effect on CD31 and VE-cadherin expression, suggesting that the elevated levels of these markers in EBs (see Fig. 3A
) resulted from an increased number of endothelial cells. Furthermore, we observed increased expression of VEGF-D in hypoxic non-endothelial cells and of VEGF-A in both hypoxic endothelial and non-endothelial cell populations (data not shown). The identity of separated cells was verified by reverse transcriptase PCR using primers against VE-cadherin and the endodermal marker 
fetoprotein (Fig. 4B
).
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During embryogenesis VEGFR-3 is initially expressed in all vasculature; later in development expression becomes restricted mainly to lymphatic vessels (31)
. To investigate expression of VEGFR-3 on developing vascular structures, EBs were cultured under normoxic or hypoxic conditions between day 8 and 12 and analyzed by immuofluorescent staining for VE-cadherin and VEGFR-3. The increased expression of VEGFR-3 induced by hypoxia was clearly localized to VE-cadherin-positive structures (Fig. 4C
). Moreover, expression of VEGFR-3 was confined to dense vascular structures in the center of the EBs. In a similar manner, EBs were analyzed by immunofluorescent staining for CD31 and the lymphatic marker Prox-1, a homeobox transcription factor involved in lymphangiogenesis during development (32)
. Staining revealed few Prox-1-positive cells mainly located in clusters, and stained cells had a punctuate morphology consistent with nuclear localization of Prox-1 (Fig. 4D
). More important, Prox-1 was not expressed in CD31-positive endothelial cells. Expression from the Prox-1 gene was also analyzed by real-time PCR. Twelve day EBs expressed relatively low levels of Prox-1 transcripts; neither hypoxia nor VEGF-A stimulation increased expression above control (Fig. 4E
). Thus, hypoxia treatment increased expression of VEGFR-3 within the vascular endothelial cell pool.
Neutralizing antibodies against VEGFR-3 blocks hypoxia-driven vascular development
The effect of hypoxia on VEGF receptor expression prompted us to investigate the contribution of these receptors to vascular development. For this purpose, EBs were cultured in hypoxia between day 8 and 12 in the absence or presence of neutralizing antibodies against VEGFR-1, -2, or -3, before being analyzed by immunohistochemical staining for CD31. In the presence of antibodies against VEGFR-1, overall vascularization increased and appeared as widespread sheets of CD31-positive cells covering most of the central part of the EB (Fig. 5
A). The VEGFR-1 antibodies also increased basal vascular structures in normoxic EBs (data not shown). In contrast, incubation with antibodies against VEGFR-2 dramatically impaired vascular network formation resulting in dispersed slender cords of endothelial cells. Treatment with neutralizing VEGFR-3 antibodies reduced vascularization almost to the same extent as VEGFR-2 inhibition. In addition, EBs treated with VEGFR-3-Ig proteins, capable of sequestering VEGFR-3 ligands VEGF-C and -D, displayed a phenotype similar to EBs treated with VEGFR-3 antibodies. Neither VEGFR-2, nor VEGFR-3 neutralization affected basal vascularization in normoxia (data not shown).
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The effect on vascularization was studied with different combinations of neutralizing antibodies. When VEGFR-1 antibodies were administered in combination with VEGFR-2 or VEGFR-3 antibodies, the inhibitory effect of the latter two was dramatically reduced (compare Fig. 5A, B
). In contrast, a combination of VEGFR-2 and VEGFR-3 antibodies resulted in complete inhibition of vascularization. Again, if VEGFR-1 antibodies were included, a small number of CD31-positive structures formed. The VEGFR-2 antibody abolished development of the peripheral vascular network in VEGF-A-treated EBs, verifying the specificity of this antibody (Fig. 5C
). Quantification of CD31-stained areas (Fig. 6
) confirmed the morphological effects seen in Fig. 5A, B
.
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In conclusion, data using neutralizing antibodies show that VEGFR-3, in addition to VEGFR-1 and -2, exerts a clear influence on vascular development and morphogenesis under conditions of low oxygen tension.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(HIF-1), the master regulator of adaptive responses to low oxygen tension (unpublished observation) (33)
. The effect of hypoxia was most pronounced during a certain stage of development, indicating that the primitive vasculature had to reach a certain degree of maturation before it could be fully remodeled by hypoxia. Therefore, this stage may resemble a postvasculogenic process, such as angiogenesis.
Low oxygen tension is a major physiological inducer of VEGF-A expression (34)
and our observed up-regulation of VEGF-A confirms an earlier report showing increased expression of VEGF-A in hypoxic EBs (35)
. Although VEGF-A is likely to have a major influence on vascular development in hypoxic EBs, the clear morphological and molecular differences between these and VEGF-A-treated EBs indicate that the effect of hypoxia also must involve other components.
The VEGF family members VEGF-C and -D have been implicated in the regulation of lymphatic endothelial cells and lymphangiogenesis (6
, 36)
. A wide body of evidence suggests that these components have angiogenic properties (6
, 36)
. In this context, the inhibitory effect of the VEGFR-3-Ig protein supports a role for VEGF-C and/or -D in EB vascularization. However, we found no evidence for activation of the Vegf-c gene in hypoxic EBs, and the contribution of VEGF-C to vascular development in our model therefore is uncertain. The VEGF-D promoter activity has recently been shown to increase under hypoxic conditions (37)
. Our data show that VEGF-D expression indeed is modulated by oxygen tension and that VEGF-D therefore is likely to mediate some of the effects induced by hypoxia. In humans, processed forms of VEGF-C and -D bind VEGFR-2 and -3, whereas in mice VEGF-D only binds VEGFR-3 (38)
. Thus, activation of the Vegf-d gene will initiate signaling in EBs exclusively via VEGFR-3 and not via other VEGF receptors, such as VEGFR-2.
In adult tissues, expression of VEGFR-3 becomes restricted mainly to lymphatic endothelial cells, but also to hematopoietic cells of monocytic lineage and to specific subsets of capillary endothelial cells (31
, 39)
. Our observed up-regulation of VEGFR-3 in hypoxic EBs did not result from an increased number of VEGFR-3-positive lymphatic endothelial cells; analysis of VEGFR-3 and Prox-1 expression revealed VEGFR-3 to be expressed on vascular endothelial cells and hypoxia to have no effect on the number of Prox-1 expressing cells at this stage. The identity of the Prox-1-positive cells is not known; these cells could represent neural progenitors as Prox-1 has been implicated in early differentiation of neural stem cells (40)
. During early embryogenesis, VEGFR-3 is expressed on vascular endothelial cells (31)
, and inactivation of the Vegfr-3 gene in mice resulted in abnormal organization of blood vessels before the emergence of lymphatic vessels (11)
. In addition, expression of VEGFR-3 seems to be up-regulated in pathological conditions characterized by neovascularization (30
, 41
42
43
44)
. These findings, combined with our data, implicate VEGFR-3 in hypoxia-driven vascularization.
The Vegfr-1 gene is transcriptionally activated in response to hypoxia (45
, 46)
. Our observation that VEGFR-1 expression is increased in hypoxic endothelial cells agrees with these findings. VEGFR-1 possesses a relatively weak tyrosine kinase activity and was initially proposed to function as a negative regulator of VEGFR-2 signaling by sequestering VEGF-A (47)
. However, some reports since then have shown VEGFR-1 activity to be important for monocyte migration, pathological angiogenesis, inflammatory responses, and protection against tissue damage (48
49
50
51
52)
. What is the function of VEGFR-1 in vascular processes? Based on several reports, it seems that VEGFR-1 activity suppresses proliferation and initiates differentiation of endothelial precursors into mature endothelial cells capable of forming organized vascular channels and tubes (53
54
55)
. It is therefore tempting to speculate that hypoxia, by increasing VEGFR-1 expression, promotes differentiation of endothelial cells and development of functional vessels. Our finding that neutralization of VEGFR-1 resulted in extensive widespread sheets of CD31-positive cells lends support for this idea.
There is now a consensus that VEGF-A exerts most of its effect on endothelial cells through VEGFR-2 (5)
. VEGFR-2 is an early marker for endothelial cells and its increased expression at E7.0 in mouse embryos correlates well with a role for this receptor in the development of endothelial precursor cells (56
, 57)
. VEGFR-2 expression has been detected at day 3 in differentiating EBs (25)
, and the importance of VEGFR-2 activity for proper embryonal vascularization was evident in our EB model. The morphological changes induced by hypoxia correlated with reduced expression of VEGFR-2. This down-regulation most likely was mediated by VEGF-A, as treatment with VEGF-A alone had the same effect. Reduced VEGFR-2 expression could be indicative of endothelial cell differentiation, since VEGFR-2 levels are down-regulated on mature endothelium (6)
.
The combined treatment with neutralizing antibodies against the three VEGFRs yielded several interesting results. Thus, neutralization of VEGFR-1 potentiated formation of immature vascular structures, compared with a variety of control conditions. This indicates that VEGFR-1 may regulate a specific subset of endothelial cells and/or that VEGFR-1 regulates certain aspects of endothelial cell biology without interference from either VEGFR-2 or -3. The most striking finding, however, was the complete inhibition of vascular development in EBs treated with both VEGFR-2 and -3 antibodies. The effect of the neutralizing VEGFR-2 and -3 antibodies alone or in combination shows that the two VEGF receptors are required for expansion and remodeling of the vascular network.
In conclusion, we show that low oxygen tension orchestrates formation of an extensive, yet morphologically unique, vascular network in which several VEGF components, notably the VEGFR-3 signaling module, fulfill an important role. These findings may provide important insights into the mechanisms underlying hypoxia-driven vascularization.
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ACKNOWLEDGMENTS |
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Received for publication February 13, 2004. Accepted for publication June 2, 2004.
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REFERENCES |
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cycle 21, respectively). ß-actin was detected at cycle 19 (N, normoxic and H, hypoxic cell populations; –RT, PCR on RNA not treated with reverse transcriptase). C) EBs were treated as in panel A and analyzed by immunofluorescent staining for VE-cadherin (red) and VEGFR-3 (green; bar 75 µm). D) EBs were cultured under hypoxic conditions between day 8 and 12 and analyzed as in panel C after staining for CD31 (red) and Prox-1 (green; bar 75 µm). E) Quantification of Prox-1 transcript levels in normoxic, hypoxic, and VEGF-A-treated EBs using real-time PCR analysis. Changes are expressed as fold induction relative to normoxic levels.
