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George M. O'Brien Kidney and Urological Disease Center, Renal Division, Departments of Medicine, Cell Biology and Physiology, and Pathology, Washington University School of Medicine, St. Louis, Missouri 63110
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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To delineate the roles of O2 and vascular endothelial growth factor (VEGF) in the process of angiogenesis from the embryonic aorta, we cultured mouse embryonic aorta explants (thoracic level to lateral vessels supplying the mesonephros and metanephros) in a three-dimensional type I collagen gel matrix. During 8 days of culture under 5% O2, but not room air, the addition of VEGF to explants stimulated the formation of CD31-positive, Flk-1-positive, Gs-IB4-positive structures in a concentration-dependent manner. Electron microscopy showed the structures to be capillary-like. VEGF-induced capillary-like structure formation was inhibited by sequestration of VEGF via addition of soluble Flt-1 fusion protein or anti-VEGF antibodies. Expression of Flk-1, but not Flt-1, was increased in embryonic aorta cultured under 5% O2 relative to room air. Our data suggest that low O2 upregulates Flk-1 expression in embryonic aorta in vitro and renders it more responsive to VEGF.
angiogenesis; embryogenesis; endothelial cell; organ culture; organogenesis; vasculogenesis
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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DURING EMBRYOGENESIS, the early vascular plexus derives from mesoderm by differentiation of vascular endothelial cells that have not yet formed a lumen (angioblasts) into primitive blood vessels. This process is termed vasculogenesis. After the primary vascular plexus is formed, more endothelial cells are generated that can form capillaries by sprouting or by intussusception from their vessel of origin in a process designated angiogenesis (11, 21).
Concurrent sprouting and nonsprouting angiogenesis are involved in the vascularization of organs such as the lung, brain, and kidney. Sprouting angiogenesis from the embryonic aorta predominates in the developing kidney (19-21).
Vascular endothelial growth factor (VEGF), produced in vascular or extravascular tissues during blood vessel formation, serves as a key endothelial growth and chemotactic agent (4, 6, 10, 11). A decrease in O2 concentration, i.e., hypoxia, activates VEGF gene and protein expression (5, 10, 11). In addition to VEGF, hypoxia induces the expression of agents that regulate VEGF gene and VEGF receptor expression, such as hypoxia-inducible factor (HIF)-1 (1, 4, 21, 26).
The finding of abnormal vascular development in the HIF-1
-deficient
mouse is strong, albeit indirect, evidence of a key role for hypoxia in
embryonic blood vessel formation (25). Supporting evidence
is provided by the observations that HIF-1 and VEGF are
spatiotemporally colocalized in regions within developing mouse embryos
in which the hypoxia marker pimonidazol can also be detected
(14).
One widely studied organ culture model of aortic-based angiogenesis employs adult mouse aortic rings from which microvessels develop in serum-free matrix culture under normoxic (17, 18), but not hypoxic, conditions (3). In contrast to what is known about the growth requirements for capillary formation from adult aortic rings, little is known about the growth of aortic rings from developing embryos (2).
To define more precisely in a model of embryonic aortic angiogenesis the roles that O2 and VEGF might play in organ vascularization, we developed a model in which formation of capillary-like structures from mouse embryonic thoracic aorta explants can be demonstrated in a three-dimensional collagen gel matrix. Low O2 and exogenous VEGF are required for angiogenesis in our explant system. Low O2 enhances the expression of the VEGF receptor Flk-1.
We propose that hypoxia and VEGF play important roles in stimulating angiogenesis from the developing thoracic aorta and that Flk-1 upregulation is one mechanism by which hypoxia renders embryonic aortic tissue more responsive to VEGF.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Preparation of three-dimensional collagen gels. Collagen gels were prepared according to the method described by Nicosia and Ottinetti (17) with some modification. Briefly, eight volumes of purified rat tail type I collagen (Sigma Chemical, St. Louis, MO) solubilized in 0.02 N acetic acid (2 mg/ml) were neutralized with one volume of premixed solution containing 200 mM HEPES, 200 mM NaHCO3, 0.05 N NaOH, and one volume of 10× MCDB-131 (Sigma Chemical). The mixture was kept on ice to preclude gelation before it was dispensed into 24-well tissue culture plates.
Embryonic aortic explant culture. Pregnant C57BL/6J mice (Jackson Laboratories, Bar Harbor, ME) were anesthetized and killed by cervical dislocation on embryonic day 14 unless otherwise specified, and the embryos were retrieved by cesarean section. Mouse embryonic dorsal aortas (thoracic level to lateral vessels supplying the mesonephros and metanephros) were visualized using an American Optical dissecting microscope, surgically dissected from embryos, and washed twice in serum-free MCDB-131 supplemented with penicillin-streptomycin. Aortic explants (~500 µm long) were sectioned and washed twice in serum-free medium. Immediately after collagen solution (300 µl) was dispensed in 24-well culture plates, the aortic explants were embedded in the collagen solution, and the plate was incubated at 37°C for 15 min to promote gelation. Some of the dissected aortas were fixed directly with zinc fixative (Pharmingen, San Diego, CA), embedded in paraffin, sectioned, and analyzed for histology.
After gelation, MCDB-131 (300 µl) supplemented with 5% heat-inactivated fetal bovine serum (GIBCO BRL, Gaithersburg, MD) was applied, and the culture was maintained at 37°C in 5% CO2-95% room air or under low (5%) O2 conditions. For low O2 conditions, explants were kept in an airtight chamber (Billups-Rothenberg, Del Mar, CA) that had been filled for 5 min at 20 l/min with 5% O2-5% CO2-90% N2. The chamber was regassed daily. Repeated measurement with a Fyrite O2 indicator (Bacharach, Pittsburgh, PA) confirmed 5% O2. The media were changed every other day. Therefore, for brief periods every 2 days, explants cultured under 5% O2 were exposed to room air. The following additions were made to cultures as indicated: recombinant human VEGF (rhVEGF), anti-human VEGF-neutralizing antibody (-hVEGF
Ab), and soluble Flt-1 (sFlt-1) fusion protein (R & D Systems,
Minneapolis, MN). Each culturing condition included at least four
cultures. Experiments were repeated at least three times.
Antibodies and histological staining reagents. Rat anti-mouse platelet endothelial cell adhesion molecule-1 (PECAM-1, also known as CD31), monoclonal antibody Mec13.3 biotin-conjugated anti-rat antibody, and FITC-conjugated anti-rat antibody were purchased from Pharmingen; anti-mouse Flk-1 and Flt-1 from Santa Cruz Biotechnology (Santa Cruz, CA); and Alexa Fluor 488-conjugated Griffonia simplicifolia isolectin IB4 (Gs-IB4) for marking endothelial cells (13) and nucleic acid staining reagents propidium iodide (PI) and Hoechst 33258 from Molecular Probes (Eugene, OR).
Western blotting analysis. Western blotting analysis was performed as described previously (23) with modifications. Briefly, aortic explants were removed from gels after 18 h of culture, rinsed with ice-cold phosphate-buffered saline (PBS), and resuspended in 80 µl of lysis buffer (1% Nonidet P-40, 20 mM Tris · HCl, pH 7.5, 10% glycerol, 1 mM leupeptin, 1 mM pepstatin A, 0.1 mM phenylmethylsulfonyl fluoride, 2 mM sodium orthovanadate). After incubation for 30 min at 4°C, insoluble materials were removed by centrifugation of cell extracts at 16,000 g for 20 min. Cell lysates normalized by protein content were placed in 5× Laemmli sample buffer, heated at 95°C for 10 min, separated by SDS-10% polyacrylamide gel electrophoresis, and electrophoretically transferred to nitrocellulose membranes (Hybond-ECL, Amersham Pharmacia Biotech, Piscataway, NJ). Identical protein loading was confirmed by staining membranes using Ponceau S dye (Sigma Chemical). The membranes were incubated for blocking by a 60-min treatment at room temperature in 10 mM Tris · HCl, pH 7.4, 100 mM NaCl, and 0.1% Tween 20 with nonfat dry milk and then immunoblotted with mouse anti-Flk-1 antibody (1:1,000) or rabbit anti-Flt-1 antibody (1:1,000; Santa Cruz Biotechnology) at 4°C. The membranes were further probed with secondary antibody, and the enhanced chemiluminescence immunoblotting detection system (Amersham Pharmacia Biotech) was used according to the manufacturer's instructions. Immunoblots were quantified using NIH Image 1.56 software. The ratio of protein expression was expressed as relative density divided by control values.
Morphological and quantitative analysis. Aortic explants, along with the collagen gels, were rinsed with ice-cold PBS twice and fixed with Bouin's solution (Sigma Chemical) for 30 min at room temperature. Samples were washed twice with PBS and treated with 1% Triton X-100 in PBS for 60 min and then incubated with 3% bovine serum albumin in PBS overnight at 4°C. After they were washed with PBS three times, samples were incubated with Gs-IB4 (20 µg/ml) or indicated antibody (primary and secondary antibody) according to the manufacturer's instructions. Gels were then sandwiched between glass slides and coverslips using GVA mounting medium (Zymed, South San Francisco, CA) and subjected to fluorescence microscopy analysis. Some samples were subjected to immunohistochemical analysis using an avidin-biotin immunoperoxidase technique (24).
Outgrowth of capillary structures was quantified by calculating the number of newly formed capillary sprouts within the cultures on a daily basis according to the method described by Nicosia and Ottinetti (17) with some modification as delineated below. Cultures were examined under bright-field microscopy using a Zeiss phase-contrast inverted microscope. The criteria used for analysis were as follows: 1) Capillary sprouts were distinguished from fibroblastic mesenchymal cells by their unique morphology (greater thickness and uniformly cohesive growth pattern). 2) The dichotomous branching of one sprout generated two new sprouts. 3) Because anastomosis between two converging vessels usually forms loop structures, each loop was counted as two sprouts (17). We confirmed that these structures were positive for Gs-IB4 staining and an endothelium-specific marker molecule PECAM-1 (7) and that there was no statistical difference between the number of capillary outgrowths determined using bright-field and fluorescence imaging. Fluorescent microscope images were captured using a Nikon Eclipse 800 microscope with a Spot 2 cooled color digital camera (Diagnostic Instruments, Sterling Heights, MI) using Spot software version 2.1. Images were imported into Adobe PhotoShop 5.0 for final processing and layout.Electron microscopy. Electron microscopy was carried out as described previously (22). Briefly, tissues were fixed in situ with 3% (wt/vol) glutaraldehyde buffer and then postfixed in OsO4. Samples were dehydrated in ethanol and embedded in Polybed 812 resin. Thin sections were stained with lead acetate and lead citrate and examined through a transmission electron microscope.
Statistics. Values are means ± SE. Data were analyzed by an analysis of variance combined with Fisher's protected least significant difference. Differences with P < 0.05 were considered to be significant.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Identification of endothelial cells within embryonic aorta.
Figure 1A is a bright-field
phase-contrast photomicrograph of a mouse aorta freshly dissected on
embryonic day 14 shown in a longitudinal view. Endothelial
cells are present at the intimal surfaces of the aortic explants, as
demonstrated by positive staining for each of the two endothelial
markers as shown in cross sections: Gs-IB4 in a
non-paraffin-embedded section shown amidst generalized nuclear PI
staining (Fig. 1B) and PECAM-1 in a paraffin-embedded section (Fig. 1C).
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Effect of hypoxia on embryonic aortic explants.
Figure 2A is a phase-contrast
photomicrograph of an embryonic day 14 mouse aortic explant
immediately after it was embedded in a collagen gel shown in
longitudinal view. If cultured under 5% O2 for 8 days, the
appearance of the explant changes in a way that suggests migration of
cells into the gel (Fig. 2B). To determine whether this is
the case, explants cultured for 2 (Fig. 2, C and F), 4 (Fig. 2, D and G), or 8 days
(Fig. 2, E and H) were stained for
Gs-IB4 without PI (Fig. 2, C-E) or
concurrently with PI (Fig. 2, F-H). A few
Gs-IB4-positive structures are detected around the aortic
explants after 2 days (Fig. 2C). As delineated by arrows, these structures stain also with PI (green + red = yellow),
consistent with a cellular identity (Fig. 2F). The number of
cells and the degree of migration increase after 6 days (Fig. 2,
D and G) and increase further after 8 days (Fig.
2, G and H).
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Effect of exogenous VEGF on embryonic aortic explants.
To ascertain whether the addition of a known mediator of angiogenesis
to explants affects their growth in vitro, aortas were cultured under
5% O2 in the presence of rhVEGF. A phase-contrast photomicrograph of an aortic explant grown for 8 days under 5% O2 in the presence of rhVEGF (50 ng/ml) is shown in Fig.
4A. Structures with a
capillary-like morphology (Fig. 4A) that stain positively for PECAM-1 (Fig. 4B) and Flk-1 (Fig. 4C) can be
delineated.
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Effect of VEGF sequestering on capillary formation from aortic
explants.
Addition of sFlt-1 to endothelial cells is thought to sequester
available VEGF and, thereby, obviate its effect (8).
Anti-VEGF-neutralizing antibody has a similar action. To ascertain
whether sequestration of VEGF affects the growth of capillary-like
structures in explants grown under 5% O2, aortas were
cultured for 8 days in the absence or presence of various
concentrations of sFlt-1 or -hVEGF Ab with or without rhVEGF (50 ng/ml).
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-hVEGF Ab has no effect on the low level of microvessels
observed in the absence of exogenous VEGF (control; Fig. 9B,
left).
As is the case for sFlt-1, addition of rhVEGF-neutralizing antibody to
explants grown in the presence of VEGF reduces the number of
microvessels to the control levels seen in the absence of rhVEGF (Fig.
9B, right).
Effect of hypoxia on VEGF receptor expression in aortic
explants.
To determine whether the action of VEGF to enhance formation of
microvessels in cultures of embryonic aorta might be mediated via
upregulation of one or more VEGF receptors, we performed Western analysis of Flk-1 and Flt-1 protein in homogenates of freshly isolated
explants (control) and explants cultured for 18 h under room air
(normoxia) or 5% O2 (hypoxia). As illustrated in Fig. 10, levels of Flk-1, but not Flt-1, are
upregulated compared with baseline when explants are grown under low
O2 conditions
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The process of blood vessel formation during development is VEGF dependent. VEGF is thought to exert actions on endothelial cells via binding to two main endothelial receptors: VEGF receptor-1 [also known as fms-like tyrosine kinase (Flt-1)] and VEGF receptor-2 [kinase insert domain-containing receptor/fetal liver kinase (Flk-1)] (1, 9). Homozygous deletions of Flt-1 or Flk-1 impair vascular development in embryos.
Hypoxia is known to be a potent stimulator of endogenous VEGF production (14, 21). Hypoxia-induced VEGF production can provide a major stimulus for the angiogenesis that accompanies organ formation. For example, in the developing retina, astrocytes that migrate together with neurons away from existing blood vessels secrete VEGF in response to the resulting hypoxia. Angiogenesis is stimulated toward the VEGF-producing astrocytes and the neurons that have accompanied them (16).
Hypoxia can also serve as a stimulus to upregulate Flt-1 and Flk-1, independent of its actions to enhance VEGF expression (16). In the case of Flk-1, the process of hypoxia-induced upregulation was demonstrated in human umbilical vein endothelial cells and in transfected porcine aortic endothelial cells. Flk-1 upregulation was reversible, maximal after 24 h of hypoxia, and regulated at a posttranscriptional level (27).
In the present studies, we use embryonic aortic explants cultured in a three-dimensional type I collagen gel matrix to show that formation of capillary-like structures is dependent on 5% O2 and addition of exogenous VEGF. Flk-1 receptors in explants grown under 5% O2 are upregulated relative to Flk-1 receptors in explants grown under room air. This observation is consistent with the basis for enhanced sensitivity to VEGF in explants grown under 5% O2 being an increase in VEGF binding to Flk-1.
In our aortic explant system, the number of microvessels formed from
aortic explants is low in the absence of added VEGF, even under 5%
O2. This is shown by direct visual comparison of Fig.
2A (no added VEGF) with Fig. 4A (VEGF added). In
the absence of VEGF, the low microvessel number is not affected by
sFlt-1 or -hVEGF Ab (Figs. 8 and 9). Our interpretation of these
data is that whatever VEGF may be produced in mouse embryonic aortic cultures or may be present in serum supplementing culture media is
insufficient to act as an effector of the angiogenic response in vitro.
It is possible that in vivo, as is the case in developing retina (16), the angiogenic stimulus for the embryonic aorta, at least the thoracic section we used for experiments, is provided by VEGF produced by nonvascular tissues outside the aorta itself. One possible source is the developing kidney (12) (see Perspectives).
Alternatively, the microvessels produced under 5% O2 could
reflect a component VEGF-independent microvessel formation or an inability of our methodology to detect a reduction by sFlt-1 or -hVEGF Ab in the setting of low levels of VEGF-stimulated
microvessel formation.
Perspectives
To our knowledge, ours is the first description of a model of angiogenesis from embryonic mouse aorta. Because we utilized thoracic aortas obtained at the level from which lateral vessels supplying the mesonephros and metanephros arise, it is possible that our model will prove useful in delineating the mechanism by which vascularization occurs in a developing organ for which angiogenesis from this part of the aorta plays a major role (the kidney).| |
ACKNOWLEDGEMENTS |
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T. Akimoto was supported by the National Kidney Foundation of Eastern Missouri and Metro East. M. R. Hammerman was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-45181 and DK-53481.
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FOOTNOTES |
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Address for reprint requests and other correspondence: M. R. Hammerman, Renal Div., Box 8126, Dept. of Medicine, Washington University School of Medicine, 660 S. Euclid Ave., St. Louis, MO 63110 (E-mail: mhammerm{at}im.wustl.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
April 11, 2002;10.1152/ajpregu.00699.2001
Received 26 November 2001; accepted in final form 11 April 2002.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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