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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We have demonstrated
that during culture under 5% O2, the addition of
recombinant human VEGF or FGF2 to mouse embryonic aorta explants
(thoracic level to lateral vessels supplying the mesonephros and
metanephros) stimulates microvessel formation. Here we show that
microvessel formation is also stimulated by addition to explants of
supernatants obtained from metanephroi grown in serum-free organ
culture or of metanephroi extracts. Supernatants and extracts from
metanephroi grown under hypoxic conditions are more stimulatory than
supernatants/extracts from metanephroi grown in room air. VEGF and FGF2
can be detected by using immunohistochemistry in developing nephrons in
the cultured renal anlagen. Metanephroi supernatants contain more VEGF
if renal anlagen are grown under hypoxic conditions than if they are
grown in room air. Metanephros supernatant-stimulated microvessel
formation is completely inhibited by soluble sFlt-1 fusion protein or
anti-VEGF antibodies (VEGF). Extract-stimulated microvessel
formation is inhibited by
VEGF or anti-FGF2 antibodies, or both. We
conclude that metanephroi produce growth factors including VEGF and FGF
that enhance microvessel formation from embryonic thoracic aorta in vitro.
embryogenesis; endothelial cell; organ culture; organogenesis
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
WE HAVE SHOWN PREVIOUSLY that during culture in a three-dimensional type I collagen gel matrix under hypoxic conditions (5% O2), but not room air (20% O2), the addition of recombinant human (rh)VEGF (2) or rhFGF2 (1) to mouse embryonic aorta explants (thoracic level to lateral vessels supplying the mesonephros and metanephros) stimulates the formation of microvessels that stain positive for the endothelial markers Griffonia simplicifolia isolectin IB4 (Gs-IB4), CD31, and Flk-1 (1, 2). Electron microscopy shows that the microvessels are capillary-like structures consisting of endothelial cells surrounding a patent lumen (2).
Growth under 5% O2 stimulates expression of the VEGF receptor (5) Flk-1 (2) and of the FGF receptor (12) FGFR1 (1) in explants. Our aortic cultures provide a model for growth factor-enhanced angiogenesis from the embryonic thoracic aorta stimulated by local hypoxia (10) within developing embryos. In vivo, one source of growth factors for the thoracic aorta might be the developing kidneys (metanephroi) (1, 2).
To determine whether chemotactic agents that are active in our model system are present in metanephroi or are secreted by metanephroi and, if so, whether they include VEGF or FGF, we 1) showed that addition of supernatants obtained from mouse metanephroi grown in serum-free organ culture or extracts of the metanephroi to mouse aortic explants stimulates microvessel formation, 2) determined that immunoreactive VEGF is present in supernatants and that immunoreactive FGF is present supernatants and extracts, and 3) showed that agents that block VEGF or FGF activity inhibit supernatant or extract-enhanced microvessel formation in vitro. We conclude that metanephroi produce growth factors, including VEGF and FGF, that enhance microvessel formation from embryonic thoracic aorta in vitro.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Embryonic tissue culture. Mouse aortas (1, 2) or mouse metanephroi (14, 16) were surgically dissected from embryos of pregnant C57BL/6J mice (Jackson Laboratories, Bar Harbor, ME) on day 14 of the pregnancy as previously described. In our hands, E14 was the earliest time at which a clearly delineated thoracic section (thoracic level to lateral vessels supplying the mesonephros and metanephros) could be dissected (1, 2).
Aortas were cultured in a three-dimensional collagen gel matrix under 5% O2 or 20% O2. Addition of 5% fetal bovine serum (FBS) to cultures is a growth requirement (1, 2). The dissected mouse metanephroi were transferred onto 0.8-µm pore size and 13-mm diameter polycarbonate sterile filter membranes (Millipore, Burlington, MA), floating on 300 µl of serum-free culture medium (MCDB-131) in 24-well culture plates, and incubated at 37°C in a humidified incubator under 5% CO2-20% O2 (designated room air; RA) or 5% O2 (designated hypoxic conditions; HC). The medium was supplemented with 1.18 g/l sodium bicarbonate, 5 µg/ml insulin, 5 µg/ml transferrin, 5 ng/ml sodium selenate, 50 ng/ml prostaglandin E1, 50 pg/ml triiodothyronine, 100 U/ml penicillin, and 100 U/ml streptomycin. Metanephroi were cultured for 4 days. The culture medium was changed on day 2. Media from the first 2 and the last 2 days of culture were stored atAntibodies and histological staining reagents. Alexa Fluor 488-conjugated Gs-IB4 for marking endothelial cells (1, 2) was purchased from Molecular Probes (Eugene, OR). Immunohistochemistry of metanephroi was performed as before (16). Anti-VEGF (SC7269) and anti-FGF2 (SC79) that recognize rat VEGF and FGF2 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) for use in immunohistochemical studies, as were control antibodies (SC2025) and (SC2027), respectively.
Morphological and quantitative analysis. Morphological and quantitative analyses of microvessel formation from aortic explants were performed as before (1, 2). Aortic explants along with the collagen gels were fixed with Bouin's solution (Sigma Chemicals) and treated with 1% Triton X-100 in PBS, followed by incubation with 3% bovine serum albumin (BSA) in PBS overnight at 4°C. Then, samples were incubated with 20 µg/ml Gs-IB4.
Outgrowth of capillary structures was quantified by calculating the number of Gs-IB4-positive capillary sprouts originating from the aortic explants in fluorescent microscope images. 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; and 3) because anastomoses between two converging vessels usually forms loop structures, each loop was counted as two sprouts. We have confirmed that these structures are positive for both Gs-IB4 staining and platelet endothelial cell adhesion molecule-1 (PECAM-1) and that there is no statistical difference between the number of microvessels determined using bright field and fluorescence imaging (1, 2).Enzyme-linked immunoadsorbent assay. Levels of mouse VEGF in supernatants of identical volumes were measured using a Quantikine mouse kit (R & D Systems). Data are expressed as picograms per milliliter.
Statistics. Experiments were performed using four sets of kidney cultures, supernatants, and extracts applied to four sets of explants on four occasions. The results are expressed as means ± SE. Data were analyzed by an analysis of variance combined with Fisher's protected least significant difference (Fisher's PLSD) test. Differences with P < 0.05 were considered to be significant.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Treatment of aortic explants with supernatants from mouse metanephros cultures. We have shown previously that the addition of 1-50 ng/ml rhVEGF (2) or rhFGF2 (1) to E14 mouse aortic explants grown under 5% O2 stimulates the formation of microvessels with a capillary-like ultrastructure over 8 days of culture. Microvessels are initially absent from explants. Microvessel formation is never observed in the presence or absence of added VEGF or FGF if explants are grown under 20% O2 (1, 2) and is minimal if explants are grown under 5% O2 without exogenous rhVEGF (2) or rhFGF2 (1).
Shown in Fig. 1A is an explant cultured under 5% O2 for 5 days in a type I collagen gel containing, as before (1, 2), control media stained with Gs-IB4. Shown in Fig. 1, B and C, respectively, are explants cultured under 5% O2 with metanephros supernatants I RA or I HC added. Shown in Fig. 1, D and E, respectively, are explants cultured under 5% O2 with supernatants II RA or II HC added. Similar to what we observed in the absence of VEGF (2) or FGF2 (1), microvessel formation is minimal in control media (Fig. 1A). However, microvessels are readily discernable in cultures to which metanephros supernatants are added (compare Fig. 1A with Fig. 1, B-E).
|
|
VEGF production and secretion from cultured metanephroi.
To gain insight into the identity of the microvessel-stimulating agent
in supernatants from developing metanephroi, we first performed
immunohistochemistry for VEGF in the metanephroi cultured for 4 days
under RA or HC. In metanephroi cultured under RA, no staining was
detected if control antibodies were substituted for the anti-VEGF (Fig.
3A). In experiments that
employed anti-VEGF, positive staining was detected (Fig.
3B). An enlargement of Fig. 3B shows that
immunoreactive VEGF was present predominantly in developing nephrons
(arrowheads) beneath the capsule within the nephrogenic zone
(15) (Fig. 3C).
|
|
|
|
Treatment of aortic explants with extracts from mouse
metanephros cultures.
Shown in Fig. 6, A and
B, respectively, are explants
cultured under 5% O2 for 5 days with metanephros extracts
RA or HC, stained with Gs-IB4. Microvessels (arrowheads)
are readily discernable in cultures to which metanephros extracts were
added.
|
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Previously, we have shown by using embryonic aortic explants cultured in a three-dimensional type I collagen gel matrix that formation of capillary-like structures is dependent on both 5% O2 and on the addition of exogenous rhVEGF or rhFGF2. The addition of 5% fetal bovine serum to cultures is an additional requirement for growth (1, 2). Therefore, growth factor effects are additive to those of serum, and the growth factors may be necessary but not sufficient to induce microvessel formation.
Flk-1 and FGFR1 receptors in explants grown under 5% O2 are upregulated relative to Flk-1 and FGFR1 receptors in explants grown under 20% O2. This observation is consistent with the basis for enhanced sensitivity to rhVEGF or rhFGF2 in explants grown under 5% O2 being an increase in VEGF binding to Flk-1 (2) or FGFR1 (1), respectively.
We postulated (1, 2) that in vivo, the angiogenic stimulus for the embryonic aorta, at least the thoracic section we used for experiments, is provided by VEGF and/or an FGF family member produced by nonvascular tissues outside of the aorta itself, one possible source being the developing kidney.
Evidence exists that both VEGF and FGFs regulate the process of embryonic kidney vascularization. In the kidney, VEGF mRNA can be detected in epithelial cells of glomeruli and in proximal and distal tubules, whereas mRNA for VEGF receptors is present in endothelial cells of glomerular and peritubular capillaries (7, 8).
A role for VEGF produced in the developing kidney as an enhancer of endothelial cell development is indicated by a number of observations. Kitamano et al. (8) showed that if endogenous VEGF activity is blocked by administration of anti-VEGF antibodies to newborn mice, blood vessel formation in the part of the kidney still undergoing development, the superficial cortex, is disturbed. Kidneys originating from antibody-treated mice had fewer nephrons than those from control animals.
Tufro (17) cocultured avascular metanephroi from E14 rat embryos with cloned and passaged mouse glomerular endothelial cells. She found that the glomerular endothelial cells invaded the metanephroi, forming capillary-like structures within and surrounding the forming nephrons. The process was accelerated and amplified by low oxygen (3% O2) and prevented by anti-VEGF neutralizing antibodies. Tufro concluded that VEGF produced by the differentiating nephrons acts as a chemoattractant providing spatial direction to developing capillaries toward forming nephrons during metanephros development (17).
Risau and Ekblom (13) reported that induced E11 and E-14-17 mouse metanephroi stimulate the growth of new blood vessels posttransplantation into the rabbit cornea. They extracted and isolated a heparin-binding growth factor from E14-17 mouse metanephroi that was mitogenic for bovine endothelial cells and stimulated angiogenesis in rabbit corneas (13). Because uninduced E11 mesenchyme had no such activity, it was suggested that differentiation imparts angiogenesis-stimulating activity to developing kidneys. Because of it size (MW~18, 000) and heparin-binding characteristics, Risau and Ekblom speculated that the endothelial mitogen was basic FGF (bFGF) (13).
Cancilla and coworkers (4) detected the expression of mRNAs for FGFs1-5, and 7-10 and FGFRs 1-4 in rat metanephroi from E14-E21 (4). Using immunohistochemistry, FGFs1 and 2 were colocalized in the ureteric epithelium, the epithelium of developing nephron elements, and the developing glomerular mesangium of metanephroi (3). FGFR1 was present in podocytes, mesangial cells, and glomerular endothelium (4). FGFR2 was detected in the lower limbs of S-shaped bodies (glomerular anlagen), and this localization was maintained in capillary loop and maturing glomeruli. In the mature-stage glomeruli, FGFR2 localization appeared to be in podocytes and mesangial cells, but not in endothelium (4).
Kloth et al. (9) examined the role of bFGF (FGF2) in renal vascular development using tissue explants prepared from kidneys of 1- to 3-day-old rabbits within the superficial cortices in which nephrogenesis is ongoing. When bFGF alone was applied to explants, vessels could no longer be detected. However, the inhibitory influence of bFGF could be overcome by the addition of VEGF or hormones such as retinoic acid and aldosterone/1,25 dihydroxyvitamin D3. Retinoic acid by itself initiated a broadening of vessel-like structures in explants. The combination of retinoic acid plus bFGF resulted in formation of considerably thinner vessels and the formation of an additional generation of endothelial structures. It was concluded that bFGF has a morphogenic rather than a mitogenic function on vascular development during kidney formation (9).
Here we show that 1) microvessel stimulatory activity in
supernatants correlates with levels of VEGF (II HC > II RA and I HC > 1 RA), and 2) supernatant-stimulated,
capillary-like structure formation can be completely prevented by
addition of sFlt-1 or mVEGF to cultures. These observations provide
strong evidence that VEGF produced in metanephroi and released into the
media is the agent responsible for the stimulation.
The renal artery arises during embryogenesis from lateral branches of the abdominal aorta that terminates in a plexus of arteries in close proximity to the renal pelvis, the renal artery rete. Persistence of one branch of this plexus results in a solitary renal artery that enters and subsequently divides within the kidney. On occasion, more than one branch may persist and there are two or more renal arteries (11).
Supernatants from metanephroi stimulate microvessel formation from
every part along the circumference of aortic anlagen. In this regard,
the enhancement is nondirectional. One possible extrapolation of our
organ culture findings to renal vascularization is that VEGF secreted
by the developing kidney is a chemoattractant for blood vessels
originating from the embryonic aorta and that directionality in vivo is
provided by the relative positions of the developing aorta and
developing kidney in the hypoxic milieu that characterizes embryonic
development (10). If such is the case, then FGF from metanephroi does not act in the same way as VEGF, because hFGF2 does
not block the microvessel formation stimulated by metanephros supernatants in vitro.
Extract-enhanced microvessel formation is inhibited by mVEGF and
hFGF2. We propose that the activity in extracts inhibited by
mVEGF reflects intracellular VEGF.
FGF2 is also present in metanephroi (Fig. 8), and the ability of
metanephros extracts to stimulate microvessel formation can be
inhibited by hFGF2. These findings are consistent with a role for an
FGF family member as a stimulator of microvessel formation.
Because it lacks a consensus signal peptide sequence (12), it is possible that, in contrast to VEGF, FGF2 or related FGFs have an impact on the process of renal vascularization once physical contact has been made between branches of the renal artery rete and the extracellular matrix of the developing metanephros (11). Alternatively or in addition, the effect of rhFGF on aortic explants in vitro (1) may reflect an action on endothelial cells that originates within the metanephros itself in vivo (6).
![]() |
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 (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.
First published February 26, 2003;10.1152/ajpcell.00436.2002
Received 20 September 2002; accepted in final form 14 February 2003.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Akimoto, T,
and
Hammerman MR.
Fibroblast growth factor 2 promotes microvessel formation from mouse embryonic aorta.
Am J Physiol Cell Physiol
284:
C378-C388,
2003
2.
Akimoto, T,
Liapis H,
and
Hammerman MR.
Microvessel formation from mouse embryonic aortic explants is oxygen and VEGF dependent.
Am J Physiol Regul Integr Comp Physiol
283:
R487-R495,
2002
3.
Cancilla, B,
Cauchi J,
Key B,
Nurcombe V,
Alcorn D,
and
Bertram JF.
Immunolocalization of fibroblast growth factors-1 and -2 in the embryonic rat kidney.
Nephrologie
2:
167-175,
1996.
4.
Cancilla, B,
Ford-Perriss MD,
and
Bertram J.
Expression of fibroblast growth factors and fibroblast growth factor receptors in the developing rat kidney.
Kidney Int
56:
2025-2039,
1999[ISI][Medline].
5.
Ferrara, N,
and
Davis-Smith T.
The biology of vascular endothelial growth factor.
Endocr Rev
18:
4-25,
1997
6.
Hyink, DP,
Tucker DC,
St. John PL,
Leardkamolkarn V,
Accavitti MA,
Abrass CK,
and
Abrahamson DR.
Endogenous origin of glomerular endothelial and mesangial cells in grafts of embryonic kidneys.
Am J Physiol Renal Fluid Electrolyte Physiol
270:
F886-F889,
1996
7.
Kanellis, J,
Fraser S,
Katerelos M,
and
Power DA.
Vascular endothelial growth factor is a survival factor for renal tubular epithelial cells.
Am J Physiol Renal Physiol
278:
F905-F915,
2000
8.
Kitamano, Y,
Tokunaga H,
and
Tomita H.
Vascular endothelial growth factor is an essential molecule for mouse kidney development: glomerulogenesis and nephrogenesis.
J Clin Invest
99:
2351-2357,
1997
9.
Kloth, S,
Gerdes J,
Wanke C,
and
Minuth WW.
Basic fibroblast growth factor is a morphogenic modulator in kidney vessel development.
Kidney Int
53:
970-978,
1998[ISI][Medline].
10.
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].
11.
Netter, FH.
Anatomy structure and embryology.
In: The Netter Collection of Medical Illustrations, vol. 6, Kidneys, Ureter and Bladder, edited by Becker EL,
and Churg J.. Pittsburgh, PA: Novartis, 1997, p. 2-35.
12.
Powers, CJ,
McClesky SW,
and
Wellstein A.
Fibroblast growth factors, their receptors and signaling.
Endocrine-Related Cancer
7:
165-197,
2000
13.
Risau, W,
and
Ekblom P.
Production of a heparin-binding angiogenesis factor by the embryonic kidney.
J Cell Biol
103:
1101-1107,
1986[Abstract].
14.
Rogers, SA,
and
Hammerman MR.
Transplantation of rat metanephroi into mice.
Am J Physiol Regul Integr Comp Physiol
280:
R1865-R1869,
2001
15.
Saxen, L,
and
Sariola H.
Early organogenesis of the kidney.
Pediatr Nephrol
1:
385-392,
1987[ISI][Medline].
16.
Sorenson, CM,
Rogers SA,
Korsmeyer SJ,
and
Hammerman Fulminant MR.
Metanephric apoptosis and abnormal kidney development in bcl-2-deficient mice.
Am J Physiol Renal Fluid Electrolyte Physiol
268:
F73-F81,
1995
17.
Tufro, A.
VEGF spatially directs angiogenesis during metanephric development in vitro.
Dev Biol
227:
558-566,
2000[ISI][Medline].
18.
Villaschi, S,
and
Nicosia RF.
Angiogenic role of endogenous basic fibroblast growth factor released by rat aorta after injury.
Am J Pathol
143:
181-190,
1993[Abstract].
|
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Visit Other APS Journals Online |