Published online before print May 24, 2001, doi: 10.1161/hh1101.091191
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© 2001 American Heart Association, Inc.
Molecular Medicine |
Vascular Endothelial Growth Factor and Basic Fibroblast Growth Factor Differentially Modulate Early Postnatal Coronary Angiogenesis
From the Department of Anatomy and Cell Biology and The Cardiovascular Center (R.J.T., A.S., W.Z., J.S.H.), University of Iowa, Iowa City, and Texas Biotechnology Corp (T.B., R.J.B.), Houston, Tex.
Correspondence to Robert J. Tomanek, PhD, Department of Anatomy and Cell Biology, Bowen Science Building, University of Iowa, Iowa City, IA 52242. E-mail robert-tomanek{at}uiowa.edu
Abstract
Abstract—The roles of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF [FGF-2]) in early postnatal regulation of coronary angiogenesis were investigated by administering neutralizing antibodies to these growth factors between postnatal days 5 and 12. Immunohistochemistry and Western blotting both revealed decreases in VEGF protein in the hearts of rats treated with either antibody. In contrast, bFGF mRNA increased in both treated groups, whereas VEGF mRNA was unchanged. Using stereological assessment of perfusion-fixed hearts, we found that both anti-VEGF and anti-bFGF inhibited the rapid and marked capillary growth that occurs during this time period and that the effects of the two neutralizing antibodies are not additive. Arteriolar growth, as indicated by a lower length density, was inhibited by anti-bFGF, but not anti-VEGF. When both anti-VEGF and anti-bFGF were administered, arteriolar length density was not significantly lower, but the population of small arterioles (<15 µm) was markedly reduced, whereas the percentage of large arterioles (26 to 50 µm) more than doubled. Thus, inhibition of both growth factors negated or limited the formation of small arterioles and facilitated an expansion of the largest arterioles. These in vivo data are the first to document that during the early neonatal period, (1) both VEGF and bFGF modulate capillary growth, (2) bFGF facilitates arteriolar growth, and (3) the two growth factors interact to establish the normal hierarchy of the arteriolar tree.
Key Words: capillaries • arterioles • myocardium • coronary circulation • development
The early postnatal period is characterized by dramatic growth of the coronary vascular bed. For example, in the rat, aggregate ventricular capillary length more than doubles between postnatal days 5 and 11.1 In humans, the number of terminal arterioles (<20 µm) increases steadily during the last 2 months of gestation and during the first postnatal year.2 Previous work in our laboratory has shown that vascular endothelial growth factor (VEGF) is highly expressed in the rat myocardium during the prenatal and early postnatal periods.3 We also documented two peaks of basic fibroblast growth factor (bFGF [FGF-2]) mRNA expression, ie, at the earliest period of embryonic capillary formation and during the early postnatal period.4 Accordingly, both growth factors are likely to play a major role in myocardial angiogenesis during this period of time. However, the conditions under which they constitute an important stimulus are not clear. Both have been found to stimulate capillary formation and growth in the embryonic chicken heart when injected in ovo.5 However, the timing of their expression during development is an important consideration, given that the effectiveness of bFGF and VEGF in increasing angioblast numbers and facilitating tube formation, respectively, in cultured embryonic hearts depends on the embryonic time point.6
That myocardial growth factors may be triggered by mechanical or metabolic stimuli has been documented in several studies.3 For example, ischemia and/or hypoxia and mechanical stretch of the myocardium have been shown to enhance VEGF expression. In thyroxine-treated rats that undergo a marked myocardial angiogenesis, bFGF mRNA is elevated. We recently tested the hypothesis that myocardial angiogenesis that occurs in response to chronic bradycardia is dependent on VEGF.7 In that study, we documented that the 23% increase in capillary growth during a 3-week treatment with the bradycardia drug alinidine was associated with 2-fold increases in VEGF mRNA and protein. In contrast, bFGF mRNA was not significantly enhanced. Most importantly, this marked angiogenesis was completely negated by administration of VEGF-neutralizing antibodies. Upregulation of VEGF also occurs in response to both static and cyclic stretch.8 9
In consideration of the above, we concluded that both VEGF and bFGF may promote the marked myocardial angiogenesis during the early postnatal growth period. We tested this hypothesis by administering neutralizing monoclonal antibodies to neonatal rats during the second week of postnatal life, during which time myocardial growth and capillary formation is so dramatic. Our studies addressed the growth of both capillaries and arterioles to test the hypothesis that VEGF and bFGF selectively regulate the formation and/or growth of these vessels.
Materials and Methods
Experimental Protocol
Five litters of Sprague-Dawley rats were divided
randomly into five groups. All protocols were approved by the Animal
Care and Use Committee of the University of Iowa and conform to Public
Health Service guidelines. Three groups received
intraperitoneal injections of VEGF or bFGF
monoclonal neutralizing antibodies or nonimmune IgG according to the
protocols previously
published.7 10
Both monoclonal neutralizing antibodies, which were prepared by us,
have been previously characterized and their specificity and
inhibitory potency described in
detail.11 12 The
antibodies were also tested for cross-reactivity; we found that no
cross-reactivity occurred. The fourth group received injections of both
neutralizing antibodies. Each rat was injected at postnatal days 5, 7,
9, and 11. The doses of neutralizing antibodies were 300 µg/100 g on
day 5 and 600 µg/100 g on the other 3 days. These rats were studied
at day 12, at which time the hearts either were fixed by vascular
perfusion with a glutaraldehyde
mixture13 or
paraformaldehyde or were rapidly frozen for protein and
mRNA analysis. The glutaraldehyde-fixed hearts
were postfixed in osmium tetroxide and embedded in Spurr’s plastic,
and 1-µm sections were prepared and stained with Richardson’s
solution. These sections were used for stereological analysis
of the myocardial vascular bed. Ultrathin sections were prepared from
selected tissue blocks, stained with uranyl acetate and lead citrate,
and viewed with a Hitachii 7000 electron microscope.
Paraformaldehyde-fixed hearts were embedded in
paraffin, and 6-µm sections from these hearts were used for
immunohistochemistry. To assess the magnitude of growth between 5 and
12 days, the fifth group of rats was studied at postnatal day 5. Hearts
from these rats were perfused with glutaraldehyde and
used for stereological analysis of the vascular
bed.
RNA Isolation and Northern Blot
Analysis
After removal of the atria, the heart was frozen in
liquid nitrogen, and total RNA was extracted using the RNA isolation
reagent RNA STAT-60 (Tel-Test Inc) according to
the manufacturer’s instructions. Northern blot analysis was
performed according to the method previously
detailed.7 We used 10 µg of
total RNA, separated on 1.2% formaldehyde-agarose gel, which was then
transferred to a Nytran Supercharge nylon transfer membrane (Schleicher
and Schuell). Hybridization of the filters was performed for 16 to 20
hours by adding VEGF, bFGF, or 18S RNA probes labeled with
[
-32P]dUTP to the
solution.
Western Blot Analysis for Growth Factor
Protein
Hearts were homogenized in cold PBS
containing protease inhibitors, and the extracted proteins
(50 µg) were run on a 15% SDS-PAGE gel. Proteins were transferred to
nitrocellulose membranes and blocked with 5% nonfat milk for 1 hour at
room temperature. We incubated the blots with VEGF rabbit polyclonal
IgG (1:500) and with bFGF rabbit polyclonal IgG (1:500) in 1% milk and
0.05% Tris-buffered saline–Tween 20.
Anti-rabbit IgG–horseradish peroxidase (1:5000) and the enhanced
chemiluminescence detection system (Amersham)
were used to visualize the antigen-antibody complexes. All antibodies
were purchased from Santa Cruz Biotechnology. Optical density of the
bands was determined using the Photoshop program.
Immunohistochemistry
Sections 6 µm thick from
paraformaldehyde-fixed and paraffin-embedded hearts
were cut and affixed to Superfrost-Plus slides. The immunohistochemical
procedures for bFGF4 and
VEGF3 have been previously
described. Endogenous peroxidases were quenched with 0.3%
H2O2, and background
staining was blocked by incubation in 5% normal bovine serum. Primary
antibody incubations were carried out in a humidified chamber, and the
tissues were subsequently incubated with a biotinylated secondary
antibody with an avidin-biotin complex (Vector
Laboratories) and then reacted with
3,3'-diaminobenzidine–H2O2.
The antibodies for VEGF and bFGF were purchased from Santa Cruz
Biotechnology. Three hearts from each group were used for histochemical
analysis.
Antibody Radiolabeling
Neutralizing antibodies were radiolabeled with
Na-125I using the iodobead-lactoperoxidase
method as previously
described.14 Rat pups were
injected with the same amount of radiolabeled antibodies described in
the Experimental Protocol section, representing
1.1x106 cpm/µg.
Capillary and Arteriolar Analysis Using
Stereology
One-micrometer sections of left
ventricular samples embedded in Spurr’s plastic were
prepared, placed on glass slides, and stained with Richardson’s
solution (Azure II and methylene blue). Capillary outlines were traced
by projecting images of the tissues, mounted on glass slides, onto
drawing paper at a magnification of x1440. The fields from each heart
included a total of 300 to 400 capillary profiles. The outlines were
scanned into a computer programmed for the assessment of various
capillary parameters (Image Pro software) as previously
detailed.5 13
Length density
(LV),
ie, total capillary length in 1 mm3
myocardium, was calculated from the long
(a) and short
(b) axes and capillary
numerical density
(NA)
using the following formula:
LV
(mm/mm3)=(a/b)NA.
Length density provides the best indicator of growth of the vascular
tree. This parameter represents the aggregate
vessel length in a unit volume of tissue and, unlike numerical density,
is not affected by plane of sectioning. Capillary surface density
(Sv) was
calculated from total perimeters
(P) of all capillary profiles
in the field as follows:
Sv=P/At,
where At
is the test or planar area of fields.
Arteriolar length densities were calculated as described for capillaries. For this assessment, we systematically scanned four tissue sections from each heart in their entirety and photographed all arterioles. These images, along with outlines of the tissue sections, determined by tracings from a microprojector, were scanned into a computer and the numerical analysis was conducted. One-micrometer sections facilitate the identification of arterioles. These vessels were identified according to the criterion that they contained at least one continuous layer of smooth muscle. An average of 121 arterioles per group (range=91 to 141) was included in one analysis. The combined sample fields from each rat averaged 2.4 mm2.
Statistical Analysis
A one-way ANOVA followed by a Student
t test and the Bonferroni
adjustment for multiple comparisons was used to analyze the
data. Statistical significance was assumed when
P
0.05. All data are
presented as
mean±SEM.
Results
Delivery of Neutralizing Monoclonal Antibodies
to the Heart
We determined radioactivity of
125I-labeled neutralizing monoclonal
antibodies in hearts 1 to 5 hours after
intraperitoneal injection. Each rat pup (10 g body
weight) was injected with 60 µg of antibody representing
6.7x106 counts of radioactivity/min.
Five hours after injection, radioactivity counts/min averaged
2.6x105 for the two antibodies. Thus,
233 ng of the antibody had accumulated in the heart by 5 hours. This
amount is 0.45% of the total injected and 6 times higher than that
expected by nonselective diffusion across the body. To remove
blood from the tissue, we perfused the heart with PBS buffer before
counting radioactivity.
Body and Heart Growth and Myocardial
Morphology
In control rats, body weight increased 83% between 5
and 12 days, whereas ventricular weight increased by 62%.
Treatment with neutralizing antibodies to either bFGF or VEGF did not
alter growth, as indicated by similar body and heart weights in these
groups and the 12-day controls. Ventricular weights (mg,
mean±SEM) in the 12-day-old rats were the following: control, 110±9;
anti-VEGF, 114±7; anti-bFGF, 113±8; and anti-VEFG+anti-bFGF, 121±10.
Histological examination of 1-µm-thick heart sections
did not reveal any differences between the treated groups and the
12-day controls. The normal morphology of the hearts from the treated
rats was verified by electron microscopy. Cardiac myocytes and the
extracellular compartments in the treated groups resembled their
counterparts in the controls.
Antibody Treatment Decreases VEGF
Protein
Immunohistochemistry for VEGF and bFGF protein is
illustrated in the micrographs in
Figure 1
. In sections from hearts of 12-day sham-injected
rats, cardiac myocytes show a strong reactivity for both VEGF and bFGF
antibodies. Anti-VEGF treatment markedly decreased immunoreactivity for
VEGF and slightly decreased immunoreactivity for bFGF. In sections from
hearts of rats treated with anti-bFGF, a slight reduction in
immunoreactivity for bFGF was observed focally in some sections. The
reduction in VEGF protein after anti-VEGF treatment was
consistent in all hearts evaluated by Western blotting
(Figure 2). In rats treated with both neutralizing
antibodies, immunoreactivity for VEGF was decreased and appeared
similar to that of rats treated with anti-VEGF alone; a slight focal
reduction in bFGF immunoreactivity was noted (data not
shown).
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Anti-VEGF and Anti-bFGF Treatment Enhances bFGF
mRNA
Expression of bFGF mRNA was enhanced 54% by anti-VEGF
and 72% by anti-bFGF treatment
(Figure 3). In contrast, VEGF mRNA was not significantly
altered by either treatment.
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VEGF and bFGF Regulate Capillary
Angiogenesis
The growth of the capillary bed was quantified by image
analysis of tissue sections from the left ventricle. Because
capillary length density tended to be higher (although this difference
was not statistically significant) in the 12-day than in the 5-day
controls
(Figure 4, top), whereas heart mass increased by 61% during
this 7-day period, one can appreciate that about one third of the
capillary bed was formed during this period of time. VEGF- and
bFGF-neutralizing antibodies significantly inhibited capillary growth,
given that capillary length density was only 82% and 83%,
respectively, of the controls. Mean (±SEM) capillary surface density
(mm2/mm3) was
also significantly lower in the anti-VEGF (10.6±0.8) and anti-bFGF
(11.2±0.5) groups than in the control group (13.4±4). Mean capillary
diameter
(Figure 4, bottom) was similar in the 5-day and 12-day
controls and in anti-VEGF and anti-bFGF groups. However, treatment with
a combination of anti-VEGF and anti-bFGF affected a significant
increase in capillary diameter. Note that capillary diameter was
5.32±0.09 in the group treated with both neutralizing antibodies,
whereas mean capillary diameter ranged between 4.70 and 4.73 in the
other 12-day-old groups (anti-VEGF, anti-bFGF, and
control).
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Capillary Morphology During Decreased VEGF and
bFGF
We used transmission electron microscopy to
quantitatively evaluate the myocardial microvasculature. Systematic
comparisons of micrographs from the treated and control groups did not
reveal differences in vascular morphology. High-magnification electron
micrographs
(Figure 5) document the normal ultrastructural
characteristics of capillary endothelial cells. Some
maturation of endothelium, primarily an increase in
vesicles, between 5 and 12 days was noted
(Figures 5A and 5B). Neutralizing antibody treatment
(Figures 5C and 5D) did not affect this maturation. In all of
the groups studied (data not shown), we consistently and
frequently observed endothelial cell processes
extending into the lumen and forming partitions. This form of capillary
proliferation was first described in
1991.15
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Arteriolar Formation and Growth
Arteriolar length density (summarized in
Figure 6, top) was not altered by normal growth, nor was it
affected by anti-VEGF treatment. In contrast, anti-bFGF treatment
lowered length density by 42%. Mean arteriolar diameter was not
altered by either treatment
(Figure 6, bottom). However, in rats administered both
neutralizing antibodies, arteriolar length density was not
significantly reduced, but mean arteriolar diameter was larger by 50%
than the 12-day-old controls
(Figure 6). Thus, simultaneous inhibition of both
growth factors affects alterations not predicted by inhibition of
either growth factor alone. Histograms illustrating the percentage of
arterioles within a diameter size class
(Figure 7, top) indicated a greater percentage of the
smallest arterioles (<10 µm) in the 5-day than in the 12-day rats.
Thus, arteriolar growth involves not only lengthening of the arteriolar
tree but diameter increases in the smallest arterioles. Inhibition of
VEGF or bFGF with neutralizing monoclonal antibodies did not notably
alter the distribution of arteriolar diameters
(Figure 7, bottom). In contrast, simultaneous
inhibition of both growth factors markedly affected a redistribution of
arteriolar diameters as illustrated in
Figure 8. This dual treatment reduced the percentage of
arterioles <10 µm by 78% and increased the largest class of
arterioles (26 to 50 µm) 2.5-fold. This shift in arteriolar diameters
is consistent with the 50% increase in mean arteriolar
diameter seen in
Figure 6 and noted above.
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The increase in coronary arteriolar diameter in the rats treated with both anti-bFGF and anti-VEGF did not affect wall thickness. Mean wall/lumen ratios (±SD) for arterioles 25 to 50 µm are the following: control, 0.83±0.02, and anti-VEGF+anti-bFGF, 0.09±0.02. For arterioles <25 µm the values are the following: control, 0.12±0.03, and anti-VEGF+anti-bFGF, 0.12±0.04.
Discussion
Our data are the first to document the reliance of myocardial angiogenesis on VEGF and bFGF during the early postnatal period. Moreover, this study provides the first in vivo data on the role and interactions of these growth factors in coronary arteriolar formation and growth. Several salient findings from this study are noted. First, capillary growth during early postnatal life is dependent on both VEGF and bFGF. Second, bFGF facilitates coronary arteriolar formation. Third, VEGF and bFGF interact in determining the hierarchy of the coronary arteriolar tree.
Treatment Protocol
VEGF and its receptors play key roles during embryonic
blood vessel development. Mice lacking one VEGF allele die during
embryogenesis.16 Embryonic
lethality also occurs in flt-1 or flk-1/KDR knockout
mice.17 18 That
the monoclonal antibodies used in this study did not attenuate heart or
body growth is consistent with the finding that newborn mice
treated with VEGF polyclonal antiserum are not
growth-restricted.19 That
study documented an inhibition of glomerular development.
In contrast, inactivation of VEGF via Cre-loxP–mediated VEGF gene
ablation or administration of the VEGF receptor chimeric protein
mFlt(1–3)–IgG in newborn mice resulted in nearly complete inhibition
of somatic growth and lethal metabolic
failure.20 This
dependence on VEGF in mice was lost after the fourth week of postnatal
life. Given that we did not find that administration of neutralizing
monoclonal antibodies to VEGF or bFGF or a combination of the two
inhibited increases in heart and body mass, the antiangiogenic effects
of the antibodies are not secondary to attenuation of somatic growth.
The doses and protocol of VEGF-neutralizing antibodies were identical
to those used in our previous
work,7 in which
bradycardia-induced myocardial angiogenesis in young adult rats was
completely blocked. Similarly, capillary growth during
revascularization in the ischemic mouse
hindlimb was totally inhibited by VEGF-neutralizing antibodies in the
same doses used in our
study.10
Our 125I labeling of each of the two neutralizing monoclonal antibodies documented their presence in the heart. The finding that VEGF protein was reduced in rats treated for 1 week with VEGF-neutralizing antibodies fits with our mRNA data indicating no upregulation for this growth factor. We suggest that the loss of VEGF protein was a consequence of its release from cells without adequate neosynthesis. In contrast, bFGF mRNA was increased by anti-VEGF as well as by anti-bFGF treatment, and as a consequence bFGF protein was not noted by Western blotting, although some slight focal decreases were noted by immunohistochemistry.
Capillary Angiogenesis
In this study we focused on the early postnatal growth
period when heart and body growth is rapid and
proportional.21 A
calculation of ventricular volume from mean
ventricular weights (ventricular
volume=ventricular weight/1.06) allows the estimation of
total capillary length. On the basis of this formula, we estimate that
ventricular volume increased from 64 to 104
mm3 between postnatal days 5 and 12. We
estimate that total capillary length (capillary length
densityxventricular volume) was 588 and 1041 m in
the 5- and 12-day control groups, respectively. Thus, 453
m, nearly one-half, of the capillary bed was formed during this
7-day period. This is consistent with an earlier
study.1 Because in the
anti-VEGF and anti-bFGF groups only 310 and 304 m were formed, one
can appreciate the magnitude of angiogenesis inhibition by the two
monoclonal antibodies. Previous work from our laboratory documented
VEGF as an essential molecule for myocardial angiogenesis induced by
chronic bradycardia in young adult
rats.7 Because bFGF mRNA was
not upregulated in that study, we concluded that it is unlikely to be a
major stimulus of growth in bradycardia-induced angiogenesis or to play
a permissive role. The current study suggests that angiogenesis in the
neonatal heart is regulated by both VEGF and bFGF. These data are
consistent with our previous in situ experiments that revealed
a strong expression of mRNA for both of these growth factors during the
early postnatal
period.3 4
Our evaluation with electron microscopy revealed a continuation of endothelial cell maturation between postnatal days 5 and 12, which is most evident during gestation.22 Because a decrease in VEGF or bFGF did not alter endothelial cell ultrastructural characteristics, nor did it affect any morphological changes in other cells in the ventricles, the antiangiogenic effects of the neutralizing antibodies were not secondary to damage due to the treatment protocol.
bFGF is clearly an angiogenic molecule as shown by both in vitro and in vivo experiments23 and is important for vessel assembly in the early embryo.24 Endothelial cells from mouse heart capillaries express both low-affinity heparan sulfate bFGF binding sites and high-affinity tyrosine kinase receptors on their surfaces, and they upregulate urokinase-type plasminogen activator and form capillary tubes in response to bFGF.25 Our finding that anti-bFGF treatment limited myocardial angiogenesis supports an angiogenic role of this growth factor during the early postnatal period. However, we recognize that bFGF may exert its influence on angiogenesis by facilitating the action of VEGF, given that VEGF expression increases when recombinant bFGF is added to cultured endothelial cells or when endogenous bFGF is upregulated.26 Moreover, bFGF has been found to induce expression of the VEGF receptor KDR (flk) via a protein kinase C and p44/p42 mitogen-activated pathway.27
Arteriolar Formation and Growth
Coronary arterioles and arteries develop when
smooth muscle cells are recruited by endothelial
cell–lined tubes or capillary-like
channels.4 28 29
The 62% increase in ventricular weight between 5 and 12
days of postnatal life noted in our study in the absence of a
significant decline in arteriolar length density indicates that a
substantial portion of the arteriolar tree was formed during this
period of time. In 5-day-old rats, 66% of the arterioles were 15 µm
or less in diameter. Seven days later, a much larger ventricle had 56%
of its arterioles in this size class. Thus, one can recognize that a
substantial number of arterioles were formed during this period. At the
same time, remodeling of arterioles upstream from the smallest branches
must have occurred to preserve the normal arteriolar hierarchy or to
increase mean arteriolar diameter. Coronary arteriolar growth
and remodeling have been documented in a porcine model of right
ventricular
hypertrophy.30
That study provided evidence of newly formed small parallel arteriolar
vessels. The fact that length density was 42% lower in the anti-bFGF
group compared with the controls suggests that arteriolar growth was
severely curtailed. Despite this growth inhibition, the arteriolar
hierarchy was not notably altered. Thus, anti-bFGF inhibited the
overall growth of the arteriolar tree rather than preventing the
formation of arterioles, as would be indicated by a reduction in the
percentage of arterioles <15 µm. Recent work has shown that FGF-1,
or acidic FGF, plays an important role in coronary artery
growth.31 Transgenic mice
with specific myocardial overexpression of the KFGF-1 gene have
increased numerical density of coronary arteries, especially
arterioles, as a consequence of increased branching. Thus, another
member of the FGF family also contributes to coronary
arteriolar formation.
When both anti-bFGF and VEGF were administered, the arteriolar hierarchy was clearly altered, whereas length density was not significantly reduced. Although the number of small arteriole profiles was markedly reduced, an inhibition of arteriolar formation cannot necessarily be assumed, because length density was not lower in the treated group. An alternative explanation is that newly formed arterioles expand rapidly and therefore contribute to the diameter classes >20 µm. Thus, in the group treated with anti-bFGF plus anti-VEGF antibodies, 56% of the arterioles are >20 µm in diameter compared with 26% in the control group. These data precipitate the conclusion that bFGF and VEGF working in concert facilitate the establishment of the hierarchy of the coronary arteriolar tree. Thus, the role of bFGF in arteriolar formation appears to overlap with that of VEGF, given that inhibition of VEGF alone does not affect arteriolar hierarchy or arteriolar length density. The finding that mean arteriolar diameter is increased when two growth factors are inhibited is consistent with our finding in capillaries. Whether other growth factors are stimulated when bFGF and VEGF are inhibited remains to be determined. The physiological consequences of the diameter changes are not clear. One outcome could be a redistribution of vascular resistances in the arteriolar bed. However, in the absence of 3-dimensional data, firm conclusions are not warranted. The major intent of this study was to determine the roles of bFGF and VEGF in arteriolar growth. The consequences of the changes attributed to decreases in these growth factors over longer periods of time are important issues for future studies.
In summary, our experiments provide new evidence that, during the early postnatal period, both VEGF and bFGF modulate coronary capillary angiogenesis, and arteriolar growth is dependent on bFGF. Our data also suggest that VEGF plays some supportive role for bFGF in arteriolar neoformation and in establishing the hierarchy of the coronary arteriolar tree.>
Acknowledgments
This work was supported by US Public Health Service Grants HL 62178, HL 48961, and DK 25295.
Footnotes
Original received October 10, 2000; resubmission received March 21, 2001; accepted April 6, 2001.
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