Fibroblast growth factor 2 promotes microvessel formation from mouse embryonic aorta

Tetsu Akimoto and Marc R. Hammerman

George M. O'Brien Kidney and Urological Disease Center, Renal Division, Departments of Medicine, Cell Biology, and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

To delineate the roles that oxygen and fibroblast growth factors (FGFs) play 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 FGF2 to explants stimulated the formation of Gs-IB4-positive, CD31-positive, and Flk-1-positive microvessels in a concentration-dependent manner. FGF2-stimulated microvessel formation was inhibited by sequestration of FGF2 via addition of soluble FGF receptor (FGFR) chimera protein or anti-FGF2 antibodies. FGFR1 and FGFR2 were present on explants. Levels of FGFR1, but not FGFR2, were increased in embryonic aorta cultured under 5% O2 relative to room air. Our data suggest that low oxygen upregulates FGFR1 expression in embryonic aorta in vitro and renders it more responsive to FGF2.

angiogenesis; embryogenesis; endothelial cell; organ culture


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ANGIOGENESIS, THE FORMATION of new blood vessels by sprouting or by intussusception from preexisting vessels, is a fundamental process for the vascularization of organs such as brain and kidney during development (3, 12, 16).

Studies employing isolated endothelial cells have defined stimulatory actions of several growth factors on blood vessel formation, including vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF2) (14, 16).

FGF2 is a chemotactic factor for endothelial cells and can promote endothelial cell differentiation in vitro (14). Among the four FGF receptors (FGFRs), FGFR1 and FGFR2 bind FGF2 with the highest affinities (15). Mice deficient for FGF2 do not manifest abnormalities of angiogenesis, at least during development (4, 11). Therefore, the wide range of angiogenic activities of FGF2 in vitro does not necessarily translate into FGF2 being the natural effector of these processes in vivo. FGF2 could mimic the action of another member of the FGF family, or more than one FGF could act on the same receptor in vivo. Whatever the case, FGF2 has been widely employed in vitro to help delineate the roles that FGFs play in vascular development (4, 11, 14, 16).

Recently, using mouse embryonic aortic explants cultured in a three-dimensional collagen gel matrix as a source of vasoformative endothelial cells, we developed a new model of angiogenesis during development (1). In our model, formation of microvessels, the ultrastructure of which reveals cells characteristic of mouse vascular endothelium surrounding an open lumen, requires that explants be grown under 5% O2 (low oxygen) and that an angiogenic stimulator (VEGF) be added to cultures (1).

The aim of the present study was to determine whether another growth factor known to be angiogenic in vitro, FGF2, promotes microvessel formation from mouse embryonic aortic explants. Here we report that microvessel formation is enhanced by FGF2 added to explant cultures grown under 5% O2 but not under room air. FGFR1 and FGFR2 are expressed in explants. Upregulation of FGFR1 observed in explants grown under 5% O2 relative to those grown under room air could render embryonic aortic explants sensitive to exogenous FGFs.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Embryonic aortic explant culture in type I collagen gel. Dorsal aortas (thoracic level to lateral vessels supplying the meso/metanephros) were surgically dissected from embryonic C57/Bl6J mice (Jackson Laboratories, Bar Harbor, ME) on day 14 (E14), sectioned, and cultured in type I collagen gels as before (1). Cultures were maintained at 37°C in a 5% CO2-20% O2 mix (room air) or under 5% O2 (hypoxic conditions) (1).

The following additions were made to cultures when indicated in the text: recombinant human FGF2 (rhFGF2), anti-human-FGF2 neutralizing antibody (alpha hFGF2-Ab), or soluble FGFR 1beta (IIIc)/Fc chimera protein [sFGF-R1(IIIc)] (R&D Systems, Minneapolis, MN). Each culturing condition included at least four cultures. Experiments were repeated at least three times.

Immunohistochemistry, antibodies, and histological staining reagents. Immunohistochemistry was performed as before (1). Rat anti-mouse platelet endothelial cell adhesion molecule-1 (PECAM-1, or CD31) was purchased from Pharmingen (San Diego, CA). Anti-FGFR1, anti-FGFR2, and anti-Flk-1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Alexa Fluor 488-conjugated Griffonia simplicifolia isolectin IB4 (Gs-IB4) for marking endothelial cells (6) and propidium iodide (PI) were purchased from Molecular Probes (Eugene, OR). No staining was observed if the primary antibody was omitted or if a species-specific IgG was substituted for it.

Western blotting analysis. Western analysis was performed as before (1). Identical protein loading was confirmed by staining membranes using Ponceau S dye (Sigma, St. Louis, MO). Membranes were blotted with rabbit anti-FGFR1 or anti-FGFR2 antibody (1:1,000) and probed with secondary antibody, followed by detection using the enhanced chemiluminescence (ECL) system (Amersham Pharmacia Biotech, Piscataway, NJ). Immunoblots were quantified using NIH image 1.56 software, and protein expression is expressed as relative density divided by control values.

Morphological and quantitative analysis. Morphological and quantitative analysis of microvessel formation from aortic explants (10) was performed as described (1) with some modifications. Aortic explants together with collagen gels were fixed with Bouin's solution (Sigma) and treated with 1% Triton X-100 in phosphate-buffered saline (PBS), followed by incubation with 3% bovine serum albumin (BSA) in PBS overnight at 4°C. Samples were then incubated with Gs-IB4 (20 µg/ml) or indicated antibody (primary and secondary antibody) combined with nuclear staining using PI. Capillary structures were quantified by counting Gs-IB4-positive sprouts originating from the aortic explants in a fluorescent microscope image (1).

Statistics. Results are expressed as means ± SE. Data were analyzed by an analysis of variance combined with Fisher's protected PI. Differences with P < 0.05 were considered to be significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Treatment of aortic explants with FGF2. We have shown that if mouse embryonic aortic explants are cultured under 5% O2, GS-IB4-positive cells originating from the explants migrate into type I collagen gels. Addition of VEGF to cultures grown under 5% O2 stimulates microvessel formation from the explants. Neither migration of cells nor microvessel formation occurs if explants are grown under room air ± VEGF (1).

To ascertain whether the addition of a known mediator of angiogenesis to explants other than VEGF enhances their growth in vitro, aortas were cultured under 5% O2 or room air in the absence or presence of rhFGF2.

Figure 1 shows explants grown under 5% O2 stained with endothelial cell markers Gs-IB4 (A-C), anti-CD31 (D), or anti-Flk-1 (E). Shown in Fig. 1A is an explant immediately after placement into a type I collagen gel. Figure 1B shows an explant after 8 days grown in the absence of rhFGF2. As before (1), Gs-IB4-positive cell migration into the gel is observed at 8 days.


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Fig. 1.   Photomicrographs of aortic explant immediately after placement in a collagen gel (A), after 8 days of growth in culture under 5% O2 (B), or after 8 days of growth in culture under 5% O2 and 10 ng/ml recombinant human fibroblast growth factor 2 (rhFGF2) (C-E). Explants are stained with Griffonia simplicifolia isolectin IB4 (Gs-IB4) (A-C), anti-CD31 (D), or anti-Flk-1 (E). Arrowheads delineate microvessels (C-E). Magnification is shown in A.

Figure 1, C-E, shows explants grown for 8 days in the presence of 10 ng/m rhFGF2. As was the case for VEGF, the addition of rhFGF2 to mouse embryonic aortic cultures grown under 5% O2 stimulates the growth of microvessels (Fig. 1, C-E).

Several concentrations of rhFGF2 were added to aortic explants grown under 5% O2, and microvessel growth was quantitated. Shown in Fig. 2 is the number of microvessels as a function of days in organ culture. On day 0, no microvessels were observed under any conditions. Under all conditions (including control), more microvessels were present on day 8 than on day 0 (P < 0.01).


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Fig. 2.   Number of microvessels in explant cultures grown for 0, 4, 6, or 8 days under 5% O2 in the absence of rhFGF2 (control) or in the presence of 1, 5, 10, 25, or 50 ng/ml rhFGF2. Data are means ± SE of 4 independent experiments. ** P < 0.01 FGF2 > control on day 8; dagger dagger P < 0.01 5 ng/ml FGF2 > 1 ng/ml FGF2, 10 ng/ml FGF2 > 5 ng/ml FGF2.

After 8 days of culture, the addition of rhFGF2 (1-25 ng/ml) to explants stimulated microvessel formation relative to the addition of no rhFGF2 (control). The number of microvessels after 8 days is significantly higher in explants grown in 5 ng/ml rhFGF2 than in explants grown in 1 ng/ml rhFGF2 and in explants grown in 10 ng/ml rhFGF2 than in explants grown in 5 ng/ml rhFGF2. Numbers of microvessels in explants grown in 10, 25, or 50 ng/ml rhFGF2 do not differ significantly from each other.

Figure 3 shows explants grown under room air stained with endothelial cell markers Gs-IB4 (A-C), anti-CD31 (D), or anti-Flk-1 (E). Shown in Fig. 3A is an explant immediately after placement into a type I collagen gel. Figure 3B shows an explant grown in the absence of rhFGF2 for 8 days. Figure 3, C-E, shows explants grown for 8 days in the presence of 50 ng/ml rhFGF2. Addition of FGF2 to explants grown under room air resulted in neither cell migration (compare Fig. 3, C-E, to Fig. 1B) nor microvessel formation (compare Fig. 3, C-E, to Fig. 1, C-E). Explants cultured in the presence of 10 or 25 ng/ml FGF2 for 8 days were identical in appearance to the explants shown in Fig. 3, C-E (50 ng/ml).


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Fig. 3.   Photomicrographs of aortic explants immediately after placement in a collagen gel (A), after 8 days of growth in culture under room air (B), or after 8 days of growth in culture under room air and 50 ng/ml rhFGF2 (C-E). Explants are stained with Gs-IB4 (A-C), anti-CD31 (D), or anti-Flk-1 (E). Magnification is shown in A.

Effect of FGF2 sequestering on capillary formation from aortic explants. We next determined the effect of FGF2 sequestration on the growth of microvessels using alpha hFGF2-Ab or sFGF-R1(IIIc) (13) to sequester added FGF2. Aortic explants were cultured under 5% O2 for 8 days in the presence or absence of varying concentrations of alpha hFGF2-Ab or sFGF-R2 with or without 10 ng/ml rhFGF2.

In the absence of FGF2, the number of microvessels was low and the addition of neither 50 ug/ml alpha hFGF2-Ab nor 400 ng/ml sFGF-R1(IIIc) changed the number (Fig. 4, A and B, first two columns). The addition of 10 ng/ml rhFGF2 to explants increased the number of microvessels. Further addition alpha hFGF2 to explants reduced the number of microvessels to the control levels seen in the absence of rhFGF2 in a concentration-dependent manner (Fig. 4A). Similarly, addition of sFGF-R1(IIIc) diminished microvessel formation from aortic explants (Fig. 4B).


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Fig. 4.   Effect of FGF2 sequestering on number of microvessels formed in aortic explants cultured for 8 days under 5% O2 in the absence (0) or presence of 10 ng/ml rhFGF2. A: effect of anti-human-FGF2 neutralizing antibody (alpha hFGF2-Ab). B: effect of soluble FGFR 1beta (IIIc) [sFGF-R1(IIIc)/Fc chimera protein]. ** P < 0.01 FGF2 + sequestering agent < FGF2 alone. dagger dagger P < 0.01 FGF2 ± sequestering agent > control.

FGFR expression in aortic explants. We next determined whether FGF receptors FGFRl and FGFR2 are present within the aortic explants. Shown in Fig. 5A is an aortic explant grown for 8 days under 5% 02 in the presence of 10 ng/ml rhFGF2 stained with Gs-IB4. Shown in Fig. 5B is the same explant stained with anti-FGFR1. An overlay (Gs-IB4 and anti-FGFR1) is illustrated in Fig. 5C. The overlay shows that FGFR1 (green + red = yellow/orange) is present in explants and microvessels (arrows).


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Fig. 5.   Photomicrographs of aortic explant after 8 days of growth in culture under 5% O2 and 10 ng/ml rhFGF2. Explants are stained with Gs-IB4 (A and C) and/or anti-FGFR1 (B and C). Arrows delineate microvessels. Magnification is shown in A.

Shown in Fig. 6A is an aortic explant grown for 8 days under 5% 02 in the presence of 10 ng/ml rhFGF2 stained with Gs-IB4. Shown in Fig. 6B is the same explant stained with anti-FGFR2. As was the case for FGFR1, an overlay (Fig. 6C) demonstrates that FGFR2 is in explants and microvessels (arrows).


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Fig. 6.   Photomicrographs of aortic explant after 8 days of growth in culture under 5% O2 and/or 10 ng/ml rhFGF2. Explants are stained with Gs-IB4 (A and C) and/or anti-FGFR2 (B and C). Arrows delineate microvessels. Magnification is shown in A.

To delineate whether the action of rhFGF2 to enhance formation of microvessels in cultures of embryonic aorta grown under 5% O2 might be mediated via the upregulation of FGFR, we analyzed the change in expression of FGFR1 and FGFR2 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. 7, levels of FGFRl, but not FGFR2, are upregulated compared with control when explants are grown under 5% O2 conditions.


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Fig. 7.   Western analysis of FGFR1 and FGFR2 in freshly isolated mouse embryonic aortas (control) and 18 h postculture under room air (normoxia) or 5% O2 (hypoxia). Data in bottom panels are means ± SE of 4 experiments ** P < 0.05 vs. control.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We have shown previously, using our mouse embryonic aortic explant model, that the addition of exogenous VEGF to cultures grown under 5% O2 (hypoxic conditions) stimulates microvessel formation (1). The present study demonstrates that microvessel formation under hypoxic conditions can also be stimulated by FGF2.

Actions of FGF2 (9, 14, 15) are mediated via signaling through cell surface receptors, which constitute a family of four structurally related transmembrane type tyrosine kinases (15). At least two of them (FGFR1 and FGFR2) are known to be expressed in endothelial cells (9, 20).

To determine whether FGF signaling plays a role in vascular development during embryogenesis, Lee et al. (9) expressed a dominant-negative mutant of FGFR1 in the developing endothelium of an E9 mouse embryo. Overexpression of the dominant-negative FRFR disrupted embryonic and extraembryonic vascular development and prevented the formation of a mature vascular network. These findings are consistent with an important role for the FGFR in the development and maintenance of a mature vascular network in the embryo (9).

Hypoxia, a known regulator of vascular development during embryogenesis (8), has been shown to upregulate high-affinity FGFRs in bovine capillary endothelial cells grown in culture (19). In cultured retinal pigment epithelium, hypoxia was shown to "prime" cells to proliferate more vigorously in response to FGF2 by increasing the cell surface FGF receptor density (5).

In the present study, we show that formation of capillary-like structures is enhanced by FGF2, but only if explants are grown under 5% O2. FGFR1 in explants grown under 5% O2 is upregulated relative to FGFR1 in explants grown under room air (Fig. 7). We have previously shown a similar hypoxia-induced upregulation of the VEGF receptor Flk-1 in the aortic explants (1).

The data shown in Fig. 7 are consistent with the basis for enhanced sensitivity to FGF2 in explants grown under 5% O2 being an increase in FGF2 binding to FGFR1, similar to the case in cultured retinal epithelium (5).

The number of microvessels formed from aortic explants is low in the absence of added FGF2 even if explants are grown under 5% O2, and in the absence of FGF2, the low number is not affected by sFGF-R1(IIIc) or alpha hFGF2-Ab (Fig. 4). Our interpretation of these data is that whatever FGFs may be produced in mouse embryonic aortic cultures or present in serum-supplementing culture media are insufficient to act as effectors of the angiogenic response in vitro.

Hypoxia is known to be a potent stimulator of FGF2 production in nonendothelial cells [rat cortical neurons (17) and human breast carcinoma cells (7)] in vitro. It is possible that in vivo, the angiogenic stimulus for the embryonic aorta, at least the thoracic section we used for experiments, is provided by FGF2 produced by nonvascular tissues outside of the aorta itself (2, 18). One candidate would be the developing mesonephros in which FGF2 is expressed only postinduction, during the time in which angiogenesis takes place (18).


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

Address for reprint requests and other correspondence: M. R. Hammerman, Renal Division, Box 8126, Dept. of Medicine, Washington Univ. School of Medicine, 660 S. Euclid Ave., St. Louis, MO 63110 (Email; 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.

First published September 25, 2002;10.1152/ajpcell.00193.2002

Received 24 April 2002; accepted in final form 19 September 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Akimoto, T, Liapis H, and Hammerman MR. Microvessel formation from mouse embryonic aortic explants is oxygen and VEGF dependent. Am J Physiol Renal Physiol 283: F487-F495, 2002.

2.   Cancilla, B, Ford-Perriss MD, and Bertram JF. Expression and localization of fibroblast growth factors and fibroblast growth factor receptors in the developing rat kidney. Kidney Int 56: 2025-2039, 1999[ISI][Medline].

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5.   Khalig, A, Jarvis-Evans J, McLeod D, and Boulton M. Oxygen modulates the response of the retinal pigment epithelium to basic growth factor and epidermal growth factor by receptor regulation. Invest Opthalmol Vis Sci 37: 436-443, 1996[Abstract].

6.   Laitinen, L. Griffonia simplicifolia lectins bind specifically to endothelial cells and epithelial cells in mouse tissue. Histochem J 19: 225-234, 1987[ISI][Medline].

7.   Lee, YJ, and Corry PM. Hypoxia-induced FGF2 gene expression is mediated through the JNK signal transduction pathway. Mol Cell Biochem 202: 1-8, 1999[ISI][Medline].

8.   Lee, YM, Jeong CH, Koo SY, Son MJ, Song HS, Bae SK, Raleigh JA, Chung HY, Yoo MA, and Kim KW. Determination of hypoxic region by hypoxia marker in developing mouse embryos in vivo: a possible signal for vessel development. Dev Dyn 220: 175-186, 2001[ISI][Medline].

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14.   Poole, TJ, Finkelstein EB, and Cox CM. The role of FGF and VEGF in angioblast induction and migration during vascular development. Dev Dyn 220: 1-17, 2001[ISI][Medline].

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17.   Sakaki, T, Yamada K, Otsuki H, Yuguchi T, Kohmura E, and Hayakawa T. Brief exposure to hypoxia induces FGF2 mRNA and protein and protects rat cortical neurons from prolonged hypoxic stress. Neurosci Res 23: 289-296, 1995[ISI][Medline].

18.   Savage, MP, and Fallon JF. FGF-2 mRNA and its antisense message are expressed in a developmentally specific manner in the chick limb bud and mesonephros. Dev Dyn 202: 343-353, 1995[ISI][Medline].

19.   Shreeniwas, R, Ogawa S, Cozzolino F, Torsia G, Braunstein N, Butura C, Brett J, Lieberman HB, Furie MB, Joseph-Silverstein J, and Stern D. Macrovascular and microvascular endothelium during long-term hypoxia: alteration in cell growth, monolayer permeability, and cell surface coagulant properties. J Cell Physiol 146: 8-17, 1991[ISI][Medline].

20.   Tsou, R, and Isik FF. Integrin activation is required for VEGF and FGF receptor protein presence on human microvascular endothelial cells. Mol Cell Biol 224: 81-89, 2001.


Am J Physiol Cell Physiol 284(2):C371-C377
0363-6143/03 $5.00 Copyright © 2003 the American Physiological Society




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