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Keywords:

  • morphogenesis;
  • vasculogenesis;
  • angiogenesis;
  • angioblast;
  • endothelial cell;
  • in vitro assay

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Here we investigated the importance of vascular endothelial growth factor (VEGF) signaling to the de novo formation of embryonic blood vessels, vasculogenesis, as opposed to the maintenance of blood vessels. We found that antagonizing the activity of the VEGF signaling pathway by using soluble VEGF receptor 1 (sFlt1) or VEGF antibodies inhibited vasculogenesis that occurs in embryos and in cultures of 7.5 days postcoitus prevascular mesoderm. Antagonist treatment resulted in the formation of clusters of endothelial cells not normally observed during vasculogenesis. In contrast, when embryos with established vasculatures or cultures of vascularized mesoderm were treated with sFlt1 or VEGF antibodies, no discernible alterations to the preexisting blood vessels were observed. These observations indicate that, although VEGF signaling is required to promote the mesenchymal to epithelial transition by which angioblasts assemble into nascent endothelial tubes, it is not required by endothelial cells to maintain their organization as an endothelium. © 2002 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Two distinct neovascular processes mediate new blood vessel formation, vasculogenesis and angiogenesis. Vasculogenesis is the de novo formation of blood vessels from mesoderm, whereas angiogenesis is the formation of blood vessels from endothelial cells (ECs) of preexisting vessels. The principle steps in the process of embryonic vasculogenesis include the generation of endothelial progenitor cells (angioblasts) from mesoderm and the transition of these mesenchymal cells to an epithelium composed of endothelial cells.

Vascular endothelial growth factor (VEGF) plays a prominent role in vasculogenesis. Mice deficient in the expression of a single VEGF-A allele exhibit failed vascular development, resulting in embryonic lethality between 11 and 12 days postcoitus (dpc; Carmeliet et al., 1996; Ferrara et al., 1996). The vital importance of VEGF signaling is further illustrated by the early embryonic lethality (8.5–9.5 dpc) and lack of blood vessel formation observed in VEGF receptor 2 (Flk1) -deficient mice (Shalaby et al., 1995). Due to the early death of these mice, it has been difficult to define the exact point in vasculogenesis impacted by deficiency of VEGF signaling. Previous studies demonstrated that VEGF signaling was not required for the generation of angioblasts from mesoderm (Drake et al., 2000). These findings suggested that VEGF signaling was important either for promoting endothelial cell behavior involved in the assembly of nascent ECs into primitive vessels or in the maintenance of primitive blood vessel integrity. Here we studied the importance of VEGF signaling to each of these processes.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Vascular Morphogenesis in the Developing Allantois

The murine allantois is an outcropping of extraembryonic splanchnic mesoderm that participates in the formation of the chorioallantoic placenta. The first blood vessels that arise in the allantois are generated by vasculogenesis (Downs et al., 1998; Drake and Fleming, 2000). Allantoic vasculogenesis is initiated at 6.5–7.5 dpc with the appearance of cells (angioblasts) that coexpress TAL1 and Flk1 (Drake and Fleming, 2000). Isolated cells expressing both proteins were detected (Fig. 1). TAL1 staining was localized to the nucleus and in cytosolic aggregates whereas Flk1 staining was diffusely distributed throughout the cytosol and in perinuclear aggregates (Fig. 1A–C). TAL1+ cells of the 6.5–7.5 dpc allantoides lacked detectable platelet endothelial cell adhesion molecule (PECAM) expression (Fig. 1D). At 8.5 dpc, angioblasts (TAL1+ cells) were present in areas adjacent to blood vessels that now expressed PECAM (Fig. 1E,F).

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Figure 1. Angioblasts are present in pre- and postvascularized allantoic mesoderm. A–D: Laser scanning confocal microscopy (LSCM) images of 7.5 days postcoitus (dpc) allantoic mesoderm labeled with TAL1 (A), Flk1 (B), TAL1 and Flk1 (C), and PECAM (D). E,F: LSCM images of 8.5 dpc allantoic mesoderm double-immunolabeled with antibodies to TAL1 (E) and PECAM (F). Scale bars = 10 μm in A–D, 50 μm in E,F.

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Vasculogenesis in Cultures of Allantoic Mesoderm

When mesoderm from prevascularized, 7.5 dpc allantoides was cultured, blood vessels formed de novo (Fig. 2A). The cells forming the new blood vessels express the endothelial cell markers Flk1, CD34, and VE-cadherin (data not shown). Expression of these three markers in newly formed vessels is consistent with findings based on studies of in vivo murine vasculogenesis (Drake and Fleming, 2000).

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Figure 2. Blood vessels formed in mesodermal explant cultures express endothelial-specific markers, have lumens and form tight junctions. A: a PECAM labeled 7.5 days postcoitus (dpc) allantoic mesodermal explant after 24 hr of culture. B: A cross-section of a vessel in an Epon-embedded cultured 7.5 dpc allantois explant (1-μm-thick section). Transmission electron microscopy analysis (C) reveals zonula adherens junctions (arrows) in apical aspects of epithelial cells, adjacent to the vessel lumen (asterisk, lumen). Arrowhead in C points to a microvillus extending from the apical surface of a putative endothelial cell. Scale bar = 100 μm in A.

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In addition to biochemical characterization of vasculogenesis as it occurs in culture, a histologic analysis was performed. These studies verified that vessels formed in vitro had lumens (Fig. 2B). Higher magnification examination revealed the presence of zonula adherens junctions between adjacent epithelial cells (presumptive ECs; Fig. 2C). Taken together, these findings indicate that vessels formed in culture have biochemical and morphologic features consistent with vessels formed in vivo.

VEGF Antagonists Abrogate In Vitro and In Vivo Vasculogenesis

The soluble form of VEGF receptor 1, sFlt1, acts to interfere with VEGF signaling (Aiello et al., 1995; Kendall et al., 1996; Ferrara et al., 1998). When 7.5 dpc mesodermal explants were cultured for 24 hr in the presence of sFlt1, vascular network formation was inhibited (Fig. 3F) compared with nontreated 7.5 dpc cultures (Fig. 3E). Likewise, embryos microinjected with sFlt1 at the 6 somite stage and cultured for 7 hr exhibited an inhibition of blood vessel formation in those regions undergoing active vasculogenesis (compare nontreated, Fig. 3A, with sFlt1-treated, Fig. 3B). Both the in vivo and in vitro responses to sFlt1 administration were similar in that small, isolated clusters of QH1/PECAM positive cells, respectively, were observed in place of linear arrangements of ECs (Figs. 3B,F, 4B). Labeling with antibodies to the transcription factor TAL1 allowed the spatial arrangement and number of constituent ECs to be discerned in arrangements of ECs as they occur under normal conditions as well as in sFlt1-induced clusters (Fig. 4C,D). High magnification examination revealed the presence of dendritic processes in both normal arrangements of ECs and in the sFlt1-induced clusters (Fig. 4E and F, respectively).

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Figure 3. In vivo and in vitro vasculogenesis respond similarly to modulation of vascular endothelial growth factor (VEGF) signaling. A–D: QH1-labeled blood vessels from regions lateral to the midline at the level of somite 8 in 10-somite stage quail embryos 7 hr after microinjection with 25 nl of control saline (A), sFlt1 (4 μg/ml; B), VEGF165 antibody (50 μg/ml; C), or VEGF165 (1 μg/ml; D). E–H: PECAM-labeled blood vessels formed from 7.5 days postcoitus (dpc) allantois explants cultured for 24 hr in the absence of exogenous agent (E) or in the presence of sFlt1 (4 μg/ml; F), VEGF165 antibody (50 μg/ml; G), or VEGF165 (1 μg/ml; H). I–L: PECAM-labeled blood vessels formed from 8.5 dpc allantois explants cultured for 24 hr in the absence of exogenous agent (I) or in the presence of sFlt1 (4 μg/ml; J), VEGF165 antibody (50 μg/ml; K), or VEGF165 (1 μg/ml; L). Similar results as depicted in J were obtained when 8.5 dpc allantois explants were incubated with 8 μg/ml sFlt1 and cultured for 24 and 30 hr (data not shown). The data are representative of at least three experiments, each performed in triplicate. Scale bars = 100 μm in A–L.

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Figure 4. sFlt1 treatment leads to endothelial cell (EC) cluster formation. A,C: Laser scanning confocal microscopy (LSCM) images of ECs formed during normal vasculogenesis in paraxial regions of the 5-somite stage quail embryo stained with QH1 (A) and TAL1 (C). B,D: LSCM images of paraxial clusters of ECs formed in a 10-somite stage quail embryo treated with sFlt1 and stained with QH1 (B) and TAL1 (D). E,F: Linear arrangements of ECs generated during normal vasculogenesis and sFlt1-induced EC clusters, respectively. Arrowheads indicate dendritic processes emanating from ECs. The data are representative of at least three experiments, each performed in triplicate. Scale bars = 12.5 μm in A–D, 5 μm in E,F.

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Similar to the response elicited by sFlt1 treatment, embryos (Fig. 3C) and cultures (Fig. 3G) treated with VEGF antibody also exhibited altered vasculogenesis. However, although the VEGF antibody-induced phenotype shared morphologic characteristics with those observed in response to sFlt1 treatment, it was less effective in the degree to which it abrogated vasculogenesis.

VEGF Antagonists Do Not Affect Established Blood Vessels

By contrast to the results observed in cultures of 7.5 dpc mesoderm, 24-hr treatment of 8.5 dpc mesoderm with VEGF antagonist, in which blood vessels had already formed, produced no gross effects on blood vessel morphology (Fig. 3J,K). Similarly, quail embryos injected at the 6-somite stage with either sFlt1 or anti-VEGF immunoglobulin (Ig) G and cultured for 7 hr showed no gross effects on established vessels (i.e., those located in the extraembryonic regions), whereas forming vessels within the embryo proper were inhibited (Fig. 3B and C, respectively).

To evaluate the apparent lack of an in vivo response to VEGF antagonists by established vessels, we examined the influence of VEGF antagonists on aortic morphogenesis. Soluble Flt1 was injected interstitially into a region lateral to the midline at the same axial level but at successive developmental stages before (4-somite stage) and after (13-somite stage) the formation of the dorsal aortae. As shown in Figure 5A, embryos microinjected with sFlt1 at the 4-somite stage and cultured for 7 hr had a pronounced inhibitory effect on the genesis of this vessel. In contrast, embryos microinjected with sFlt1 at the 13-somite stage and cultured for 7 hr did not exhibit discernible alterations in aortic morphology (Fig. 5B). Taken together, the findings from both explant cultures and embryo microinjections suggest that formed blood vessels do not require VEGF stimuli to retain their structure.

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Figure 5. sFlt1 disrupts forming but not established dorsal aortae. A,B: Laser scanning confocal microscopy (LSCM) images of sFlt1-injected, QH1-labeled 4- and 13-somite stage quail embryos, respectively. Shown are regions located lateral to the midline and adjacent to somites 2–8 (7 hr post-sFlt1 microinjection). In contrast to B, the dorsal aorta is absent in A. No overt effects on the dorsal aorta were apparent when 13-somite stage embryos were microinjected with sFlt1 and cultured for 24 hr (data not shown). The data are representative of at least three experiments, each performed in triplicate. Scale bar = 100 μm.

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Established Blood Vessels Respond to VEGF Stimuli

Although angioblasts/ECs in 8.5 dpc cultures express Flk1 (data not shown), the lack of a response to VEGF antagonists puts into question whether the VEGF signaling pathway was intact. As VEGF is known to promote vascular hyperfusion (Drake and Little, 1995), cultures of 8.5 dpc mesoderm were treated with VEGF. As shown in Figure 3L, treatment resulted in profound alterations in vascular patterning; the avascular regions typically observed in control cultures (Fig. 3I) were replaced by ECs (PECAM+). A similar result was observed when cultures of 7.5 dpc mesoderm or 6-somite embryos were treated with VEGF (Fig. 3H and D, respectively). Analysis of thin sections from VEGF-treated cultures showed that small-caliber vessels characteristic of controls had been replaced by large vascular sinuses (data not shown). These findings establish that ECs in 8.5 dpc cultures are capable of responding to VEGF stimuli. In light of these results and those obtained by using the VEGF antagonists, we conclude that VEGF signaling machinery may not play an active role in the maintenance of an established blood vessel.

Morphometric Analysis Corroborates Visual Assessments of the Effects of Agents on In Vitro Blood Vessel Formation

In addition to visual assessment of the effects of agents on in vitro vasculogenesis, we also used quantitative morphometric techniques. Specifically, we measured the percentage of a standard optical field occupied by blood vessels (PECAM+ cells), an index that we refer to as vascular density. As shown in Table 1, analysis of 7.5 dpc cultures showed that the mean vascular density values of each treatment group (VEGF, sFlt1, or anti-VEGF) were statistically different (all P values < 0.001) when compared with the control cultures, consistent with the visual assessments described above. Within each culture group (i.e., control, sFlt1-treated, anti-VEGF–treated, and VEGF-treated), the mean vascular density values showed very little deviation.

Table 1. Mean Vascular Density Measurements of Control and Treated Allantois Explant Culturesa
dpcControlVEGFsFlt1VEGF antibody
  • a

    Values indicated represent the percentage of a standard optical field occupied by blood vessels (PECAM+ cells). Standard deviation values are denoted by ±. dpc, days postcoitus; VEGF, vascular endothelial growth factor.

  • *

    P values are > 0.05 when compared with controls. All other P values were < 0.001.

7.523.4 ± 6.3 (n = 16)57.7 ± 6.3 (n = 6)8.1 ± 3.2 (n = 7)13.3 ± 1.5 (n = 3)
8.541.2 ± 8.7 (n = 51)81.3 ± 6.3 (n = 16)39.7 ± 8.7 (n = 12)*38.8 ± 1.0 (n = 4)*

In contrast to findings from cultures of 7.5 dpc mesoderm, only vascular density values from the VEGF-treated 8.5 dpc cultures were statistically different (P values < 10−7) from controls. Vascular density values from sFlt1- and VEGF antibody-treated 8.5 dpc cultures were not statistically different from controls (Table 1), a finding consistent with visual assessments (Fig. 3J,K) that also indicated no apparent effect of these agents on vascular morphogenesis in cultures of 8.5 dpc mesoderm. Again, analysis showed little variation of values within control and treatment groups. Overall, these findings show a relatively low variability in the density of blood vessels formed during normal culture. Most importantly, agent-induced alterations in vascular density are consistent for each agent and can be statistically differentiated from controls.

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Embryonic vasculogenesis is dependent upon signal transduction involving VEGF and its receptors (Fong et al., 1995, 1999; Shalaby et al., 1995; Carmeliet et al., 1996; Ferrara et al., 1996). For example, mouse embryos deficient in the expression of VEGF or Flk1 fail to form blood vessels (Shalaby et al., 1995). A similar outcome has been observed when avian embryos were treated with the VEGF antagonist sFlt1 (Drake et al., 2000). Although dramatic, these VEGF signaling-deficient phenotypes have not led to an understanding of the specific step for which VEGF signaling is critically required in the process of vasculogenesis. Here we have addressed this question by studying the consequence of antagonizing VEGF signaling in both the quail embryo and in an in vitro mesoderm culture system. Our findings reveal heterogeneity in the response to suppression of VEGF stimuli between the processes by which blood vessels are formed and the processes by which they are maintained, specifically, the mesenchymal to epithelial transition in which angioblasts assemble into nascent endothelial tubes is inhibited by VEGF antagonists. However, the maintenance of an organized endothelium (i.e., a blood vessel) shows no obvious dependence on VEGF stimuli.

Our results suggest that there is a narrow window within the progression of blood vessel formation, specifically the period during which angioblasts transition to form nascent endothelial tubes (Fig. 6A), for which VEGF is critically required (Fig. 6A). The limits of this window correspond to the period after the “birth” of angioblasts and extend through to the formation of a lumenized blood vessel. Our specification of this window is consistent with other findings showing that mice deficient in VEGF-A (Carmeliet et al., 1996; Ferrara et al., 1996) or in the VEGF receptor Flk1 generate angioblasts (Shalaby et al., 1995) and that ECs fail to form tubes in the absence of VEGF signaling (Koolwijk et al., 2001; Yang et al., 2001).

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Figure 6. Diagram depicting the process of de novo blood vessel assembly and the consequences of suppression of vascular endothelial growth factor (VEGF) signaling. A: A diagrammatic representation of intermediates of the process of vasculogenesis, beginning with an undifferentiated mesodermal cell and culminating in the formation of a nascent blood vessel. B: Depiction of possible consequences of antagonizing VEGF signaling in which endothelial cell aggregates do not progress to an established vessel, but instead form abnormal endothelial cell clusters. C: Graphic depiction of the lack of an overt response of an established blood vessel to antagonized VEGF signaling.

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We have shown that VEGF antagonist treatment of primitive mesoderm or early embryos leads to formation of clusters of angioblasts/ECs (Fig. 6B,C). The large number of angioblasts/ECs in these clusters and their rounded morphology distinguish them from angioblast/EC aggregates observed as part of normal vasculogenesis (Drake et al., 1997). How suppression of VEGF signaling leads to the excessive clustering of angioblasts/ECs is unknown. Regulation of cell adhesive interactions represents processes that are likely dependent on VEGF signaling. The expression of VE-cadherin during normal vascular development at the precise time when angioblasts/ECs are engaged in the de novo assembly of blood vessels suggests that this molecule may be critically involved in vasculogenesis. That VEGF acts to regulate VE-cadherin expression is suggested by similarities between the effects of VEGF antagonists observed in the present study and those described in VE-cadherin–deficient mice (Gory-Faure et al., 1999). In both cases, abnormal clustering of angioblasts/ECs was associated with the failure of blood vessels to organize. Thus VEGF signaling may be critical for VE-cadherin to promote assembly of angioblasts/endothelial cells to nascent blood vessels and counteract excessive clustering. In addition to regulation of cell–cell adhesion, VEGF signaling may be critical to promote cell–matrix interactions that are associated with the changes in cell shape and motility processes that occur during early blood vessel assembly. Previously, Drake et al. (Drake et al., 2000) speculated that suppression of VEGF signaling inhibited protrusive activity of normal endothelial aggregates engaged in blood vessel assembly. In the present study, we show that sFlt1 treatment did not abrogate the ability of ECs to produce fine dendritic cellular processes (Fig. 4E,F). However, this does not preclude the possibility that formation of larger cell processes might be affected by suppression of VEGF signaling. In this regard, VEGF has been shown to induce expression of α1β1 and α2β1 integrins in ECs (Senger et al., 1997), and microinjection of integrin function-blocking antibodies leads to the inhibition of blood vessel assembly, resulting in the formation of rounded endothelial cell clusters (Drake et al., 1992, 1995) similar to what is observed in response to VEGF antagonist treatment.

In counterpoint to the profound effects on the assembly of blood vessels in response to suppression of VEGF signaling was the finding that abrogation of VEGF signaling had no overt effect on established blood vessels in the embryo. Although, as discussed below, there is precedence for this result, it was none the less surprising, because suppression of VEGF had been shown previously to lead to the regression of blood vessels that are similar to those in the early embryo. For example, hyperoxia-mediated suppression of VEGF expression led to the regression of retinal capillary blood vessels (Alon et al., 1995). Similarly, VEGF conditional suppression led to the regression of tumor vessels (Benjamin and Keshet, 1997). Although both studies indicate that the response of blood vessels varied, in the case where regression was correlated with vessel type, only the larger arteries and veins were refractory to suppressed VEGF levels (Alon et al., 1995; Benjamin and Keshet, 1997).

We conclude that the lack of vascular regression in response to VEGF antagonists was not due to deficiencies in the VEGF signaling pathway. This is supported by the fact that ECs composing nascent blood vessels of the early embryo both express VEGF receptors (Drake and Fleming, 2000) and are capable of responding to exogenous VEGF stimuli (Fig. 3L). As we observed no evidence for established blood vessel regression in response to inhibition of VEGF signaling, our findings indicate that suppression of VEGF is not in and of itself sufficient to cause vascular regression. The regression observed in the hyperoxic eye and glioma tumors (Alon et al., 1995; Benjamin and Keshet, 1997) may be due to synergy between the consequence of reduced VEGF signaling and signaling pathways associated with the hyperoxic state or tumor pathobiology, respectively.

Our findings led us to conclude that VEGF signaling is not required for the maintenance of the vascular endothelium but acts in formed vessels to regulate a more restricted subset of VEGF's known biological activities. Potential activities would include regulating vascular permeability or mediating an angiogenic response to elevated VEGF stimuli that accompanies various pathologic conditions. Such a restricted role for VEGF is consistent with the fact that hyperoxia treatment results in suppression of VEGF expression and that the conditional knockout produces no overt effects on established blood vessels (Alon et al., 1995; Benjamin and Keshet, 1997). Similarly, Gerber et al. (Gerber et al., 1999) have shown that the requirement for VEGF activity for survival diminished through early postnatal development and is not required beyond neonatal week 4. This reduction in the requirement for VEGF correlates with the transition from active neovascularization, occurring in early postnatal development to the steady state condition of the vasculature in the young adult.

Recent studies indicate that EC progenitors derived from the circulation contribute to the formation of new blood vessels associated with conditions such as prosthesis implantation, diabetic and myocardial ischemia, and wound healing (Kennedy and Weissman, 1971; Asahara et al., 1999; Schatteman et al., 2000; Kocher et al., 2001). Because VEGF antagonists inhibit the assembly of blood vessels from EC progenitors (angioblasts) in the embryo, we speculate that they may likewise inhibit assembly of blood vessels from circulating endothelial progenitors. If true, this may provide the mechanistic explanation for the ability of anti-VEGF drugs, such as sFlt1, to inhibit blood vessel formation in adult pathologic conditions.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Antibodies and Other Proteins

Rabbit anti-mouse TAL1 and rabbit anti-recombinant TAL1 was obtained from Dr. Steven Brandt (Vanderbilt University and VA Medical Center, Nashville, TN). Rat monoclonal antibodies to PECAM/CD31, VE-cadherin/CD144, and Flk1/VEGFR-2 were purchased from BD Pharmingen (San Diego, CA). Rat anti-mouse CD34 was purchased from Research Diagnostics, Inc. (Flanders, NJ). QH1 hybridoma supernatant developed by F. Dieterlen was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biological Sciences, Iowa City, IA 52242. Donkey anti-rabbit, anti-mouse, and anti-rat IgG conjugated to either fluorescein isothiocyanate or indodicarbocyanine were purchased from Jackson ImmunoResearch Labs, Inc. (West Grove, PA). Recombinant human VEGF165, sFlt1, and goat anti-VEGF165 IgG were obtained from R&D Systems (Minneapolis, MN).

Isolation and Culture of Primitive Murine Mesoderm

Adult CD-1 mice (Mus musculus; Charles River, Raleigh, NC) were used solely as a source of very early stage 7.5–8.5 dpc embryonic tissue. Adult animals were killed by Forane (isoflurane) inhalation followed by cervical dislocation. According to the Report of the AVMA Panel on Euthanasia (1993), killing by anesthesia followed by cervical dislocation is both an accepted and effective method of killing. Embryos (7.5–8.5 dpc) were dissected from mice and placed into Dulbecco's PBS (DPBS) (4°C). The allantoides, purely mesodermal structures, were then dissected away from embryos in DPBS (4°C) and transferred to wells of 4-well chamber slides (Nalgene Nunc, Naperville, IL) containing 0.4 ml of Dulbecco's modified Eagle's medium (Gibco BRL/Life Technologies, Baltimore, MD), 10% fetal bovine serum (Gibco) and 1% penicillin, streptomycin/L-glutamine (Gibco) plus or minus the indicated agonists/antagonists. The extent of dilution of the culture medium with DPBS did not exceed 0.5%. The isolated mesoderm was then cultured for 24 hr at 37°C, 5% CO2.

Confocal Microscopic Analysis of Cultures

The culture medium was infused with 0.6 ml of 3% paraformaldehyde, to achieve a concentration of 2%. Fixation proceeded for 25 min, at 25°C. Fixative was removed, and the cultures were washed twice in DPBS, 0.01% sodium azide (DPBSA). Cultures were then treated for 40 min with 0.02% Triton X-100, DPBSA to permeabilize the cells. After a 40-min blocking treatment with 3% bovine serum albumin/DPBSA, primary antibodies (10 μg/ml) in DPBSA were added to the cultures and incubated for 1 hr, at 25°C. The cultures were washed with DPBSA and fluorochrome-conjugated anti-IgG (Jackson ImmunoResearch Labs, Inc.) was added at 10 μg/ml and incubated for 1 hr. After washing with DPBSA, mounted allantois cultures were analyzed by using a Bio-Rad MRC-1024 laser scanning confocal microscope (Bio-Rad, Cambridge, MA).

Morphometric and Statistical Analysis

Vascular density, defined as the percentage of a standardized optical field (1550 μm2) occupied by PECAM-positive cells/vessels, was determined from laser scanning confocal microscopy images by using NIH Image 1.62 (black pixels representing immunolabeled vessels and white pixels representing avascular areas). Microsoft Excel was used to determine mean vascular density values and standard deviation for each group (control, VEGF, sFlt1, anti-VEGF) at each stage (7.5 or 8.5 dpc). This program was also used to perform two-way Student's t-tests for each cultured experimental group (VEGF, sFlt1, or anti-VEGF) compared with the cultured stage-matched, nontreated group. Each experiment was performed a minimum of three times to ensure statistical power.

Microinjection, Ex Ovo Culture, and Confocal Analysis of Avian Embryos

Methods for microinjection, ex ovo culture, and confocal microscopy of early stage avian embryos have been described previously (Drake and Little, 1991; Drake et al., 1992, 1997).

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

The authors thank Dr. Steven Brandt for the generous gift of rabbit anti-mouse and anti-recombinant TAL1 antibodies (Vanderbilt University and VA Medical Center, Nashville, TN). The authors also thank Dr. Debra Hazen-Martin (Medical University of South Carolina, Charleston, South Carolina) for her expert assistance with transmission electron microscopy. W.S.A. and C.J.D. received funding from the NIH, and C.J.D. was funded by the DAMD.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
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