Transcriptional Regulation of Vascular Endothelial Growth Factor Expression in Epithelial and Stromal Cells during Mouse Mammary Gland Development
Russell C. Hovey1,
Anita S. Goldhar1,
Judit Baffi and
Barbara K. Vonderhaar
Laboratory of Tumor Immunology and Biology (R.C.H., A.S.G.,
B.K.V.) National Cancer Institute and Laboratory of Immunology
(J.B.) National Eye Institute National Institutes of Health
Bethesda, Maryland 20892
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ABSTRACT
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Accompanying changes in the development and
function of the mammary gland is the establishment of a vascular
network of critical importance for lactogenesis and tumorigenesis. A
potent angiogenic and permeability factor that regulates vascular
development in association with epithelial-stromal interactions is
vascular endothelial growth factor (VEGF). Analysis of VEGF
transcription by RT-PCR revealed mRNA for all three VEGF isoforms
(VEGF120, 164, 188) within the mammary gland of
nulliparous females. During pregnancy the level of
VEGF188 declined and became undetectable during
lactation in association with the increased abundance of
VEGF120 and VEGF164
mRNAs. All three isoforms were expressed at consistent levels within
the cleared mammary fat pad throughout development. Furthermore, the
presence of VEGF188 mRNA in omental adipose
tissue at various stages established that
VEGF188 is expressed specifically in adipose
tissue within the mammary gland. Using 3T3-L1 preadipocytes it was
demonstrated that VEGF188 mRNA transcription
occurs as a late event during lipogenesis distinct from earlier
induction of VEGF120 and
VEGF164 mRNA during differentiation. In
contrast, HC11 mammary epithelial cells only expressed mRNA for
VEGF120 and VEGF164.
Localization of VEGF mRNA and protein revealed that VEGF is expressed
in stromal cells of the mammary gland in nulliparous females and then
undergoes a transition to epithelial expression during lactation. By
contrast, mRNA for the VEGF receptors, Flk-1 and Flt-1, localized to
stromal cells within the mammary fat pad during virgin and gestational
development and was expressed in the interstitial tissue basal to
epithelial cells during lactation. Taken together, these results
support the conclusion that VEGF is differentially transcribed by
specific cell types within the mammary gland, and that under hormonal
regulation it functions in an autocrine/paracrine manner.
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INTRODUCTION
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In assuming its ultimate function as an active secretory organ,
the mammary gland develops a complex vasculature during the course of
its postnatal development (1). In nulliparous females, capillaries
arise from distal arteries and ramify between adipocytes within the
mammary fat pad (2). Capillaries are characterized by fenestrations,
microvillus processes, pinocytotic vesicles, and short marginal folds
(3). During pregnancy a dense capillary plexus forms around the rapidly
proliferating alveoli, such that by parturition and during lactation
each alveolus is surrounded by a basket-like capillary plexus. During
established lactation there is considerable capillary meandering within
the mammary gland, presumably in adaptation to the active secretory
state of the alveoli (2). In pregnancy and lactation, capillary walls
become thin (2) while microvillus processes elongate (4) to facilitate
the transport of nutrients and metabolic waste (1). Furthermore, the
number of pinocytotic vesicles used for nutrient transport and the
number of marginal folds used to slow blood velocity are maximal in
late pregnancy and lactation (3).
Several key hormones regulate mammary gland growth and function, as
well as development of the mammary gland vasculature. Treatment of
ovariectomized mice with estrogen plus progesterone leads to increased
blood vessel diameter and capillary formation (1, 5). Additional
administration of PRL or adrenalcorticoids augments this effect,
although neither hormone alone has any effect (1). Furthermore,
hormone-induced capillary outgrowth is influenced by the mammary
epithelium and epithelial-stromal interactions as various hormone
combinations failed to stimulate capillary growth in the
epithelium-free mammary fat pad (1), pointing to additional control by
locally produced growth-stimulatory agents.
Vascular endothelial growth factor/vascular permeability factor
VEGF/VPF is a potent stimulator of endothelial cell proliferation,
angiogenesis, and vascular permeability (6). Several VEGF isoforms are
alternatively spliced from a single transcription start site under the
regulation of a single promoter. Three VEGF isoforms have been
described in the mouse (VEGF120,
VEGF164, and VEGF188) while
an additional isoform, VEGF206, has been
identified in humans (7, 8, 9). The two smaller mouse isoforms are soluble
and freely secreted, whereas VEGF188 is
membrane-associated (10) due to transcription of a highly basic domain
encoded by exon 6. All three VEGF isoforms have both mitogenic and
permeabilizing properties (10) as glycosylated homodimers (6, 11). VEGF
initially stimulates vascular permeability (12) with a
103-fold greater potency than histamine (13).
After longer exposure, VEGF promotes endothelial proliferation as well
as sprouting and migration (12) through the liberation of several
proteases (14, 15). The critical role for VEGF is demonstrated from
VEGF gene-ablation studies wherein heterozygous mice display defective
interconnection of embryonic vasculature and spatial organization of
endothelial cells in the vessels of the yolk sac and a reduced lumen in
the dorsal aorta (16). VEGF is produced by a wide variety of tissues
and cell types including glandular epithelial and stromal cells of the
endometrium (14), adipocytes (15), and vascular smooth muscle cells
(8). Furthermore, VEGF is produced in various adult tissues that do not
demonstrate active angiogenesis, including pituitary, brain, kidney,
lung, adrenal gland, heart, stomach mucosa, liver, and spleen (11).
Expression of VEGF is notably up-regulated in breast (17, 18) and other
tumors and correlates with poor prognosis (18).
Targets for VEGF are the two high-affinity tyrosine-kinase receptors,
Flk-1/KDR (19) and Flt-1 (20). Mouse mutants for Flk-1 lack a dorsal
aorta and vitelline artery, blood islands, and organized blood vessels
of the yolk sac (21) whereas Flt-1 mutants possess a thickened
endocardial lining and heart ventricle along with enlarged vasculature
and disorganized blood islands of the yolk sac (22). While VEGF
receptors on endothelial cells are considered the primary site of VEGF
action, other cell types, including epithelial and stromal cells from
breast, ovary, and prostate tumors (23, 24, 25), also express VEGF
receptors. Despite these observations, roles have not been ascribed to
these receptors in nonendothelial cells.
Within this study we have investigated the role for VEGF and its
receptors during normal mammary gland development in association with
significant angiogenesis and cell-cell interaction. Our findings reveal
transcriptional alteration of VEGF gene expression during normal
mammary gland development and specific expression of
VEGF188 within the mammary fat pad. Distribution
of VEGF and its receptors supports a paracrine role for VEGF during
vascularization of the mammary gland.
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RESULTS
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Adipose-Specific Transcription of VEGF188
in the Mammary Gland
To determine the ontogeny of VEGF expression within the intact
mammary gland, we analyzed VEGF mRNA transcription by RT-PCR. Primers
spanning the mouse mVEGF gene amplified the three reported mRNA
variants, VEGF120, VEGF164,
and VEGF188, as the expected 431-, 563-, and
635-bp products, respectively. During the course of development,
expression of VEGF188 declined from high levels
in the prepubescent female to low levels during pregnancy and became
undetectable during lactation, subsequently increasing during
involution (Fig. 1A
). By contrast,
abundance of both VEGF120 and
VEGF164 showed a stage- specific increase
(>2-fold) during midpregnancy and lactation. Given that the decline in
VEGF188 expression reflected a change in mammary
gland composition from predominantly adipocytes in the nulliparous
female to predominantly epithelium in lactation, we explored whether
VEGF188 mRNA expression was exclusive to the
mammary fat pad. Compared with the intact mammary gland, expression of
VEGF188 was maintained in the cleared fat pad
(CFP) during all stages of development while the abundance of
VEGF120 and VEGF164
remained consistent across all stages (Fig. 1B
).

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Figure 1. Expression of VEGF mRNA Isoforms in the Intact
Mouse Mammary Gland (A) and CFP (B) during Postnatal Development
Total RNA from intact tissue was extracted from pooled no. 4 abdominal
mammary glands from at least eight mice killed at the indicated stages
(week, wk; or day, d) of development. Mice killed at 6 weeks were at
estrus (E). For CFP tissue, endogenous epithelium was surgically
removed at 3 weeks of age, and total RNA was extracted from pooled
tissues of at least six mice killed at the indicated ages. Samples
taken at 3 weeks of age and earlier were from the epithelium-free
region of the mammary fat pad. Reverse-transcribed RNA was amplified by
PCR (28 cycles) using primers that span all VEGF isoforms, and products
revealed on agarose gels were stained with ethidium bromide.
Corresponding amplification of GAPDH from the same reverse
transcription reaction is shown.
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Development- and Tissue-Regulated Expression of VEGF Receptor
mRNA
The developmental expression of mRNA for the VEGF receptors, Flt-1
and Flk-1, was investigated within the intact mammary gland (Fig. 2
). Amplification by RT-PCR was
quantified and expressed as a function of the respective
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA level (data not
shown). Abundance of Flk-1 was elevated in the first 2 weeks of life,
declining to the lowest level at 5 weeks and subsequently increasing
during puberty to maximal expression at 12 weeks of age. By contrast,
Flt-1 mRNA expression was consistent during these phases of
development. During pregnancy the expression of both receptor mRNAs
declined to reach the lowest level around parturition. The expression
of both mRNAs increased during early lactation to peak at 710 days of
lactation, thereafter declining to low levels at the time of
weaning.

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Figure 2. Expression of Flk-1 and Flt-1 in the Intact Mammary
Gland during Normal Mammary Gland Development
Total RNA from pooled tissue samples collected at various stages (week,
wk; or day, d) of development was amplified by RT-PCR using
gene-specific primers for Flk-1 and Flt-1. Pubescent females were
killed at the indicated stages at either diestrus (Di) or estrus (E).
Amplification of GAPDH was performed on the same reverse transcription
reactions. Samples were analyzed on agarose gels stained with
ethidium-bromide.
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To determine the stromal expression of Flt-1 and Flk-1, CFP tissue was
analyzed at various stages of development (Fig. 3
). Similar to the intact mammary gland,
abundance of Flk-1 was elevated in the first 2 weeks of life,
thereafter declining to a steady state during the remainder of
development. By comparison, expression of Flt-1 mRNA within the mammary
fat pad was relatively consistent across all stages examined.

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Figure 3. Expression of Flk-1 and Flt-1 in the Cleared
Mammary Fat Pad during Mammary Gland Development
Total RNA was prepared from pooled CFP samples at the indicated stages
(week, wk; or day, d) of development as described in Fig. 1 . Pubescent
females were killed at the indicated stages at either diestrus (Di) or
estrus (E). Amplification of GAPDH was also performed on the same
reverse transcription reactions. Samples were analyzed on agarose gels
stained with ethidium-bromide.
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Localization of VEGF mRNA and Protein and VEGF Receptor mRNA within
the Developing Mammary Gland
The distribution of VEGF mRNA within the mammary gland during its
development was determined by in situ hybridization using a
35S-labeled riboprobe complementary to all
mVEGF mRNA transcripts. Within the mammary glands of nulliparous
females, VEGF mRNA primarily localized to adipocytes and connective
tissue within the mammary fat pad (Fig. 4A
), although a low level of expression
was also evident in the ductal epithelium. Similarly, VEGF mRNA
expression in midpregnancy localized to a majority of, but not all,
adipocytes within the mammary fat pad while expression was distinctly
absent from stromal fibroblasts adjacent to the mammary epithelium
(Fig. 4B
). A low level of expression was also homogeneously distributed
within the ductal epithelium whereas mRNA abundance was greater in the
newly formed alveolar buds. During lactation, VEGF mRNA was
specifically expressed in the alveolar epithelium and in greatest
abundance by cells lining less-distended alveoli (Fig. 4C
).
Furthermore, heterogeneity in the abundance of VEGF mRNA within
individual epithelial cells was evident. The distribution of VEGF mRNA
was compared with the localization of VEGF protein by
immunohistochemistry. Within the mammary gland of pregnant females,
immunoreactivity was primarily associated with the adipose and
connective tissue elements of the stroma (Fig. 5
, A and B), although isolated epithelial
cells also displayed mild VEGF immunoreactivity. In addition, nuclear
staining was detected in endothelial cells of the vasculature as well
as in endothelial cells within the supramammary lymph node (Fig. 5C
).
Macrophages were also positive for VEGF (Fig. 5C
). Within the lactating
mammary gland, VEGF immunoreactivity localized primarily to the
alveolar epithelium (Fig. 5D
). Consistent with in situ
hybridization results, protein expression was greater in epithelial
cells of less-distended alveoli compared with engorged,
secretion-filled alveoli. Furthermore, immunoreactive protein was more
concentrated toward the apical membrane within secretory epithelial
cells.

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Figure 4. Localization of VEGF mRNA within the Mammary Gland
during Postnatal Development
Sections of mammary tissue from female mice at 3 weeks of age (A), day
8 pregnancy (B), and day 10 lactation (C) were hybridized to a
35S-labeled antisense riboprobe complementary to exons 23
of VEGF mRNA. D, Serial section to panel B hybridized with sense probe.
Nuclear fast red counterstain. Scale bar for all
panels = 100 µm.
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Figure 5. Immunohistochemical Localization of VEGF within the
Mammary Gland during Development
Paraffin sections were incubated with a polyclonal anti-VEGF antibody
and immunoreactive complexes detected using nickel-intensified DAB.
Sections were not counterstained. A, Mammary gland, day 9 of pregnancy,
displaying immunoreactivity associated with connective tissue
surrounding the mammary ducts. B, VEGF immunoreactivity was also
localized to adipocytes of the mammary fat pad. C, Within the
suprammamary lymph node, heterogeneous VEGF immunoreactivity was
detected in macrophages (solid arrow) and in the
vascular endothelium (open arrow). D) Distribution of
VEGF within mammary tissue on day 10 of lactation showing localization
to the alveolar epithelium and greater immunoreactivity in distended
alveoli compared with less distended alveoli. E, Negative control.
Scale bar for all panels = 50 µm.
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Western analysis of immunoprecipitated BALB/c mammary tumor and
lactating tissue extracts was performed to identify VEGF forms present
in the mammary gland. Cytosolic and membrane fractions in both tissues
contained a predominance of VEGF164 (Fig. 6
), whereas lesser levels of
VEGF120 were detected after longer exposures (not
shown). The slightly retarded migration of
VEGF164 relative to the recombinant protein
positive controls likely represents the reported glycosylation of this
variant.

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Figure 6. Western Analysis of VEGF Protein Expressed in the
Lactating Mammary Gland and DMBA-Induced Mammary Tumor
Total protein (1 mg) was immunoprecipitated with a goat anti-mVEGF
antibody, electrophoresed, and probed with a rabbit antihuman VEGF
antibody before chemiluminescent detection. Lane 1, Recombinant human
VEGF120 (25 ng); lane 2, recombinant human
VEGF165 (25 ng); lane 3, membrane fraction from a
DMBA-induced mammary tumor (22 ); lane 4, cytoplasmic fraction from a
DMBA-induced mammary tumor; lane 5, mammary gland membrane fraction,
day 15 of lactation; lane 6, mammary gland cytoplasmic fraction, day 15
of lactation; lane 7, recombinant mVEGF120 (25 ng); lane 8,
recombinant mVEGF164 (25 ng); lane 9, negative control,
immunoprecipitated tumor cytoplasm (1 mg) without primary antibody.
Migration of molecular size markers is indicated.
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We used in situ hybridization to localize Flt-1 and Flk-1
mRNA within the mammary gland during its development. In nulliparous
females, mRNA for both receptors localized primarily to the adipose and
connective tissue stroma (Figs. 7
, A and
B, and 8, A and B). Concurrent with
alveolar development during pregnancy, the distribution of Flt-1 and
Flk-1 became more heterogeneous and punctate, localizing to distinct
sites within adipose tissue and the interstitial stromal fibroblasts
(Figs. 7C
and 8C
). Within the lactating mammary gland, mRNA for both
receptors localized basal to the alveolar epithelium (Figs. 7D
and 8D
)
and became more punctate during established lactation (Figs. 7E
and 8E
). Expression of Flk-1 mRNA was also localized to brown adipose
tissue in the lactating thoracic mammary gland (Fig. 7E
). Under these
exposure conditions it was not possible to localize whether Flt-1 or
Flk-1 mRNA was expressed by the mammary epithelium.

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Figure 7. Localization of Flk-1 mRNA during Mouse Mammary
Gland Development Using a 35S-Labeled Riboprobe Generated
from a cDNA Corresponding to the Intracellular Domain of mFlk-1
Mammary tissue was from females in the following states: A, 3 week
nulliparous; B, 8 week nulliparous; C, day 8 pregnancy; D, day 1
lactation; E, day 1 lactation showing brown fat tissue present in the
thoracic mammary glands. F, Serial section to E probed with sense
strand riboprobe. Nuclear fast red counterstain. Scale
bar for all panels = 100 µm.
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Figure 8. Localization of Flt-1 mRNA by in
Situ Hybridization in the Mouse Mammary Gland
Paraffin sections were hybridized with a 35S-labeled
riboprobe complementary to mRNA encoding the intracellular domain of
Flt-1. Mammary tissues were at the following stages: A, 3 week
nulliparous; B, 8 week nulliparous; C, day 8 pregnancy; D, day 1
lactation; E, day 10 lactation. F, Serial section to E probed with
sense strand probe. Scale bar for all panels = 100
µm. Nuclear fast red counterstain.
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Specific Transcription of VEGF188 by
Adipocytes within the Mammary Fat Pad
Given the expression of VEGF188 within the
mammary fat pad, transcription of VEGF188 was
further examined in white adipose tissue. Analysis of VEGF
transcription in omental adipose tissue revealed that
VEGF188 is transcribed at significant levels
during all stages of development (Fig. 9
). Transcription of VEGF was also
examined in 3T3-L1 preadipocytes that undergo differentiation and lipid
accumulation in vitro. During differentiation, 3T3-L1 cells
increased transcription of VEGF120 and
VEGF164 mRNA (Fig. 10
, A and B) concurrent with the
induction of aP2 gene expression, a marker of adipocyte differentiation
(Fig. 10C
). Exposure of differentiated cells to insulin and FCS led to
significant accumulation of lipid droplets (not shown) that was
paralleled by the initiation of VEGF188
transcription after 4 days of treatment and a further increase in
VEGF120 and VEGF164 mRNA
levels (Fig. 10B
). These results confirm the expression of
VEGF188 by mature, lipid-filled adipocytes.
Transcription of VEGF mRNA was also examined in undifferentiated and
differentiated HC11 mammary epithelial cells to establish the
specificity of VEGF188 expression within the
mammary gland (Fig. 10D
). Consistent with our findings in
vivo, HC11 cells expressed only mRNA encoding
VEGF120 and VEGF164,
confirming the specific transcription of VEGF188
by adipocytes within the mammary fat pad.

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Figure 9. Expression of VEGF mRNA in Female Mouse Omental
Adipose Tissue at Various Developmental Stages
Total RNA was subjected to RT-PCR using primers that span all isoforms
of mVEGF. Samples were as follows: lane 1, 5 week nulliparous female;
lane 2, day 15 gestation; lane 3, day 5 lactation; lane 4, day 5
involution. Corresponding amplification of GAPDH mRNA from the same
reverse transcription reaction is depicted.
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Figure 10. Expression of VEGF mRNA by Cultured Preadipocytes
and Mammary Epithelial Cells
3T3-L1 preadipocytes cultured in 10% FCS were differentiated for
2 days with dexamethasone plus 3-isobutyl-1-methylxanthine (IBMX) and
then cultured in 10% FCS plus insulin for 2, 4, 5, and 6 days to
promote lipid accumulation. A, Analysis of VEGF mRNAs during lipid
accumulation in 3T3-L1 cells using RT-PCR and primers spanning VEGF.
Samples were from cells at confluence (confl.), after 2 days
differentiation (diff.), or during a period of lipid accumulation. B,
Quantification of VEGF mRNA isoforms expressed during lipid
accumulation in vitro. Data are means ±
SEM (n = 3). *, P < 0.05
vs. respective level at confluence. C, Corresponding
Northern analysis of fatty acid binding protein aP2 expression during
preadipocyte differentiation and lipid accumulation. D, Analysis of
VEGF mRNA expression in HC11 mammary epithelial cells by
RT-PCR. Cells were grown to either 80% confluence (subcon) or remained
undifferentiated for 2 days in growth medium after reaching confluence
(undiff). Cells were also differentiated for 2 days after reaching
confluence using medium supplemented with PRL (1 µg/ml) and
hydrocortisone (1 µg/ml) in the absence of epidermal growth
factor.
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DISCUSSION
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Functional development of the normal mammary gland involves
a complex interaction between multiple cell types during several major
phases of reproductive development (26). Central to this development is
the establishment and maintenance of a vascular supply to support the
mammary fat pad and associated epithelial proliferation, lactogenesis,
and regression (1, 2) as well as tumor formation (18). Despite the
integral function of the vasculature within the mammary gland, little
has been reported concerning the regulation of its development during
normal mammogenesis. Here we have investigated the role of VEGF, a
potent angiogenic peptide present as several variants in multiple
tissues that acts through VEGF-specific receptors.
Within this study we identified alterations in VEGF gene
transcription during mouse mammary gland development using PCR primers
spanning all eight exons. Stage-specific VEGF transcription was
associated with the increased expression of
VEGF120 and VEGF164 mRNA
from midgestation and into lactation while the abundance of
VEGF188 declined concurrently. Sustained
expression of VEGF188 within the CFP during
concurrent states suggested that the disappearance of this transcript
during late gestation and lactation in the intact mammary gland
represented the epithelial-induced depletion of adipocytes within the
mammary fat pad (4, 27). The subsequent demonstration that
VEGF188 is abundantly expressed in omental
adipose tissue and lipid-filled 3T3-L1 cells confirmed that
VEGF188 is expressed by adipocytes of the mammary
fat pad during normal mammary development. Examples describing the
physiological regulation of VEGF188 mRNA
transcription are limited, although it has been shown that
hyperoxia-induced damage in the lung coincides with a down-regulation
of VEGF188 gene expression (28). Rat uterus
exposed to estradiol shows an increase in VEGF188
after 6 h compared with an earlier up-regulatory effect after
2 h for VEGF120 and
VEGF164 (23). In contrast, treatment of human
endometrial stromal cells with estradiol increases
VEGF121 and VEGF165
expression, while the level of VEGF189 mRNA is
unaffected (24).
The present data indicate that adipocytes and depots of white adipose
tissue such as the mammary fat pad express significant levels of mRNA
encoding all three VEGF isoforms. Along these lines, Tonello et
al. (29) recently reported the expression of all three VEGF
isoforms in brown adipose tissue of rats, consistent with our in
situ hybridization findings in brown adipose tissue that is
associated with the thoracic mammary fat pad (27). However, expression
of VEGF188 mRNA could not be induced in cultures
of isolated brown adipocytes, possibly because full differentiation was
not achieved (29). From the results of experiments using 3T3-L1
preadipocyte cells it is clear that VEGF188
expression is specifically up-regulated during late lipogenesis in
association with adipocyte hypertrophy. By contrast,
VEGF164 and VEGF120 mRNAs
increase during early differentiation, concurrent with the expression
of several early fat cell-specific genes such as aP2 (15) and PPAR-
.
While an up-regulation of VEGF188 mRNA in mature
white and brown adipose tissue suggests an association between
VEGF188 and lipid accumulation, its precise
function remains unclear. A highly basic 24-amino acid insertion
derived from exon 6 results in VEGF188 remaining
strongly cell associated (30). Interestingly, exon 6 of the VEGF gene
is homologous to exon 6 of the platelet-derived growth factor (PDGF)
A-chain that enables nuclear targeting of PDGF after cleavage of the
signal sequence (31). While VEGF188 demonstrates
similar mitogenic effects relative to the other isoforms (10), the
small amount of VEGF188 that is secreted can
induce greater vascular permeability relative to the shorter VEGF
isoforms (30). Compared with VEGF120 and
VEGF164, VEGF188 does not
diffuse beyond the tumor mass and fails to promote peripheral
vascularization (32). Up-regulation of VEGF189
has also been reported in human vascular smooth muscle cells after
phorbol ester activation (8), suggesting an involvement of AP-1
signaling. A potential function for VEGF188 mRNA
expression within adipose tissue may be to enhance vascular
permeability and subsequent uptake of triglyceride precursors to
facilitate lipogenesis. By comparison, mammary epithelial cells did not
express VEGF188 despite their requirement for
active nutrient and precursor uptake from the mammary vasculature
during lactogenesis. Hence, VEGF188 may fulfil a
specific role for cells such as adipocytes. Whether the absence of
VEGF188 expression by mammary epithelial cells
prevents a negative permeabilizing effect of this molecule on the
alveolar epithelium remains to be assessed.
Both VEGF120 and VEGF164
mRNAs are expressed in epithelial and stromal compartments of the
murine mammary gland during its development. Whereas
VEGF120 is acidic and does not bind heparin,
transcription of exon 7 to encode VEGF164 leads
to its slightly basic and heparin binding characteristics (30) that may
result in its sequestration to extracellular matrix components (33)
supporting the mammary epithelium. These two VEGF isoforms are
primarily secreted and exert mitogenic and permeabilizing effects on
vascular endothelial cells (11). In contrast to a recent report by
Pepper et al. (34), our comprehensive analysis of VEGF
expression in the mammary CFP across development by RT-PCR, combined
with in situ hybridization and immunohistochemical analyses,
indicates that the stroma is a primary source of VEGF within the
developing mammary gland. This concurs with localization of the
majority of fluorescence to the mammary stroma of transgenic mice
carrying a VEGF promoter fused to green fluorescent protein (35).
Furthermore, primary cultures of stromal cells from normal breast and
breast tumors express significant amounts of VEGF protein under normal
(36) and hypoxic (37) conditions.
Taken together, our results indicate that locally derived VEGF may
contribute to vascular development within the mammary gland and
supporting fat pad. The abundance of VEGF expression by adipocytes
during ductal development, and by adipocytes and epithelial cells
during alveolar development and lactogenesis, tentatively makes
available a considerable level of ligand within the mammary gland.
Heterogeneous distribution of Flt-1 and Flk-1 mRNA throughout adipose
tissue and the interstitial fibroblastic stroma likely associates with
the dispersed capillary endothelium that is recognized as the primary
site of VEGF action (19, 20). However, a recent report by Zangani
et al. (38) demonstrated that mammary stromal cells also
express Flk-1 and can transform to endothelial-like cells and form
microcapillaries in response to VEGF. Hence, expression of Flt-1 and
Flk-1 by adipocytes and the interstitial fibroblasts may represent a
stromal population that is recruited during neovascularization in
response to adipocyte- or epithelial-derived paracrine VEGF.
Interestingly, Flk-1 expressed by fibroblasts is a shorter molecule
than that on endothelial cells (19). What remains to be established is
the specific role, if any, for adipoctye-derived
VEGF188 vs.
VEGF120 and VEGF164 during
this development.
While not a focus of this study, VEGF also appears to fulfil a major
function within the lymphatic system of the developing mammary gland.
Expression of VEGF mRNA and the localization of VEGF protein suggest
that macrophage-synthesized or mammary gland-derived VEGF may target
vascular endothelium within the lymph node. This proposal is in keeping
with the demonstrated expression of VEGF and its receptors by
macrophages (39). Hence it is conceivable that the permeabilizing
effects of VEGF on the vascular endothelium could ultimately modulate
the migration of malignant epithelium into the circulating lymphatic
system.
The increase in VEGF120 and
VEGF164 mRNA abundance within the intact mammary
gland during late gestation and lactation reflected increased
expression in the alveolar epithelium. This finding is consistent with
the recent observations of Pepper et al. (34) showing
increased total VEGF expression during these stages. Extensive
angiogenesis occurs within the mammary gland during this period (1, 34)
to establish a complex capillary network surrounding each alveolus for
efficient delivery and transfer of water and solutes to the epithelium
(1). Consistent with a proposed paracrine role for VEGF in tissues such
as breast (36, 40) and ovarian carcinoma (25), mRNA for Flt-1 and Flk-1
localized primarily to interstitial cells basal to the alveolar
epithelium in an area of extensive neovascularization (1). A notable
observation was that VEGF mRNA and protein levels were greater in
epithelial cells of less-developed alveoli relative to fully distended
alveoli. Such heterogeneity may reflect the paracrine stimulation of
angiogenesis by epithelial cells proliferating during early lactation
(1, 2) or their ability to modulate vascular permeability (12) and
regulate nutrient supply during early lactogenesis (2). Heterogeneous
distribution of gene expression for milk proteins has also been
described within the lactating rodent mammary gland (41). Therefore, a
permeabilizing effect of VEGF would accompany the essential requirement
for increased blood flow to the mammary gland during lactation.
Furthermore, the intracellular localization of immunoreactive VEGF
within the alveolar epithelium suggests that VEGF may be secreted into
milk. Indeed, immunoreactive VEGF is present in human milk (34, 42) and
may act on the intestinal epithelium in the neonate (42).
Taken together, these results indicate multiple roles for VEGF within
the developing and functional mammary gland under the influence of
hormonal and epithelial-stromal interactions. Differential
transcription of VEGF mRNA is consistent with distinct roles for the
different VEGF isoforms in adipocytes and epithelial cells.
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MATERIALS AND METHODS
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Animals and Tissues
BALB/c mice were housed in 12 h light, 12 h darkness
with ad libitum access to food and water. Mice were used in
accordance with the NIH Guide for the Care and Use of Laboratory
Animals. Mammary tissue was collected from mice killed by cervical
dislocation at various stages of postnatal development. Females killed
in pregnancy were mated at 10 weeks of age, where the day of seminal
plug detection was considered day 0 of pregnancy. Females killed during
mammary involution were weaned after 10 days of lactation. The
supramammary lymph node was removed before snap freezing in liquid
nitrogen. In addition to mammary tissue, omental fat tissue from the
abdominal cavity was collected from females killed at specific ages.
Mammary tumor tissue was excised from a female 5-month-old BALB/c mouse
that had been treated with 9,10-dimethyl 11,2-benzanthracene (DMBA)
at 8 weeks of age.
To study changes in the expression of VEGF and VEGF receptor mRNA
within the mammary stroma, mammary fat pads were cleared of endogenous
mammary epithelium to leave a CFP according to the method of DeOme
et al. (43). Briefly, female mice at 3 weeks of age were
anesthetized with avertin and the abdominal no. 4 mammary glands were
exposed through a ventral Y-shaped incision. The lymph node and
nipple-associated tissue were cauterized to leave a CFP devoid of
endogenous epithelium. Recovered mice were subsequently euthanized at
various stages of development. Samples of CFP were obtained from
unoperated females at 3 weeks of age and younger by collecting the
epithelium-free region of mammary fat pad dorsal to the supramammary
lymph node. Mice killed in lactation nursed 68 pups from the
remaining unoperated mammary glands. CFP tissue was snap-frozen in
liquid nitrogen and all tissues were stored at -80 C.
RNA Extraction and Northern Analysis
Total RNA was extracted from tissues and cell cultures using
Trizol (Life Technologies, Inc., Gaithersburg, MD). Intact
and CFP mammary tissue was pooled from animals killed at each age.
Total RNA (5 µg) from 3T3-L1 cells for Northern analysis was
electrophoresed through 1% agarose-formaldehyde and transferred to
GeneScreen (NEN Life Science Products, Boston, MA). A
432-bp cDNA fragment corresponding to mouse aP2 (a gift from Dr.
Constantine Londos, NIDDK, NIH) was labeled by random priming using
32P-dCTP (Rediprime, Amersham Pharmacia Biotech, Buckinghamshire, U.K.) and hybridized overnight at 65
C. Stringency washes were performed to 0.5x SSC at 65 C.
Semiquantitative RT-PCR
Total RNA from mammary tissues was treated
with DNAse I (Life Technologies, Inc.) before reverse
transcription. Reverse transcription of tissue and cell culture RNA (1
µg) to first strand cDNA was performed using MMLV reverse
transcriptase (Life Technologies, Inc.) primed with oligo
dT and random hexamers in a reaction volume of 25 µl. Amplification
of the RT product (2.5 µl) by PCR was performed using PCR master mix
(Roche Molecular Biochemicals, Indianapolis, IN)
with the following primers and conditions. The three splice variants of
VEGF described in mice (VEGF120,
VEGF164, and VEGF188) were
amplified using 5'- and 3'-primers located in exons 1 and 8,
respectively.
VEGF 5'-CTG CTC TCT TGG GTC CAC TGG
VEGF 3'-CAC CGC CTT GGC TTG TCA CAT 94 C, 3 min for 1 cycle, then
94 C, 15 sec; 60 C, 30 sec; 72 C, 1 min for either 2123 cycles (HC11
and 3T3-L1 cells) or 26 cycles (mammary and adipose tissue).
Expected product sizes for VEGF120,
VEGF164, and VEGF188 were
431, 563, and 635 bp, respectively.
Flt-1 5'-AGC CCA CCT CTC TAT CCG CTG G
Flt-1 3'-GGC GCT TCC GAA TCT CTA ACG 95 C, 1 min; 57 C, 1 min; 72
C, 2 min for 25 cycles. Expected product size was 664 bp.
Flk-1 5'-AGC TTG GCT CAC AGG CAA CAT CGG
Flk-1 3'-TGG CCC GCT TAA CGG TCC GTA GG 95 C, 1 min; 60 C, 1 min;
72 C, 2 min for 25 cycles. Expected product size was 624 bp.
Amplification conditions were optimized for each experiment to utilize
a final number of PCR cycles within the linear phase of product
amplification. Levels of gene expression determined by RT-PCR were
normalized to respective mGAPDH levels using primers from
CLONTECH Laboratories, Inc. (Palo Alto, CA) amplified
under optimized conditions. Quantitative analysis was achieved by
measuring the intensity abundance of PCR products in gel photographs
using NIH Image software.
Western Analysis
Homogenized DMBA-induced mouse mammary tumor and lactating
mammary tissue (day 15 lactation) were separated into membrane and
cytosolic protein fractions by ultracentrifugation at 37,000 rpm for
1 h. Protein fractions (1 mg) were immunoprecipitated overnight at
4 C using a rabbit antihuman VEGF antibody (20 µl of P20 goat
anti-mVEGF polyclonal antibody, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) in a volume of 1 ml. Immunocomplexes were
separated using Protein A sepharose beads (Pharmacia Biotech, Uppsala, Sweden) that were collected by centrifugation
and washed with PBS. Samples were boiled in sample buffer,
electrophoresed on a 14% polyacrylamide gel under reducing conditions,
and transferred to nitrocellulose. The membrane was blocked in 5%
milk-Tris-buffered saline-0.1% Tween (TBST) for 1 h, and
then incubated with a rabbit antihuman VEGF antibody (147, 1:500;
Santa Cruz Biotechnology, Inc.) overnight at 4 C. After
washing in TBST, the blot was incubated with a donkey antirabbit
secondary antibody (1:2000, Amersham Pharmacia Biotech)
for 3 h, washed with TBST, and incubated with enhanced
chemiluminescence (ECL, Amersham Pharmacia Biotech) to
detect immunocomplexes. Negative controls, similarly immunoprecipitated
and then incubated with only secondary antibody, did not demonstrate
any specific signal. Positive controls were 25 ng each of recombinant
mVEGF120 and mVEGF164, and
25 ng each of recombinant human VEGF121 and
VEGF165 (R & D Systems, Minneapolis, MN).
In Situ Hybridization
A riboprobe complementary to all VEGF mRNA isoforms was
transcribed from a cDNA encoding exons 23 of mVEGF. Riboprobes for
Flt-1 and Flk-1 were transcribed from PCR products described above that
were subcloned into PCRscript. Templates were linearized with either
NotI or EcoRV and in vitro
transcription was performed using 35S-UTP and T3
or T7 polymerase (Ambion, Inc. Austin, TX).
Mammary tissues were fixed overnight in buffered 4% paraformaldehyde
at 4 C and then embedded in paraffin. Sections on silanized slides
(ProbeOn, Fisher Scientific, Pittsburgh, PA) were dewaxed,
pretreated in 0.2 N HCl, digested with proteinase K (15
µg/ml), and acetylated using 0.25% acetic anhydride in 0.1
M triethanolamine. After prehybridization, sections were
hybridized with cRNA probes (7 x 104
cpm/µl) overnight and then washed to 0.1x SSC followed by digestion
of nonspecifically bound probe using RNAse A (20 µg/ml). Sections
were dipped in emulsion (NTB-2, Kodak, Rochester, NY) and
exposed for 48 weeks at 4 C and then counterstained with nuclear fast
red.
Immunohistochemistry
Paraffin sections were deparaffinized with xylene, rehydrated,
rinsed in PBS before immunostaining using the ABC
(avidin-biotin-peroxidase complex) method. To block endogenous
peroxidase activity, sections were immersed in PBS containing 3%
H2O2 before blocking with
1% bovine serum containing 0.6% Triton X-100 for 1 h at room
temperature. Endogenous biotin was blocked using the biotin/avidin
blocking kit (Vector Laboratories, Inc., Burlingame, CA).
Sections were incubated with a rabbit polyclonal anti-VEGF antibody
(1:500, Santa Cruz Biotechnology, Inc.) overnight at 4 C.
Sections were rinsed with PBS and then incubated with biotinylated
antirabbit IgG (1:1,000, Vector Laboratories, Inc.) for
1 h at room temperature. Reagents from the Vectastain
Elite ABC kit (Vector Laboratories, Inc.) were used
according to the manufacturers instructions. Immunoreactivity was
detected using 0.05% DAB (diaminobenzidine) intensified with
ammonium-nickel. Sections were dehydrated and coverslipped using DePex
(Fluca Chemical Co., Buchs, Switzerland). Negative
controls were incubated in 1% bovine serum containing 0.6% Triton
X-100 instead of the primary antibody and showed a lack of
immunoreactivity.
Cell Culture
Mouse 3T3-L1 preadipocyte cells (a gift of Dr. Jaideep Moitra,
NIDDK, NIH) were routinely grown in DMEM supplemented with 10% FCS.
Cells seeded into six-well plates (2 x 105
cells per well) were grown to confluence for 2 days and then changed to
differentiation medium [10% FCS plus 0.5 mM
3-isobutyl-1-methylxanthine (IBMX, Sigma, St. Louis, MO)
and 1 µM dexamethasone] for an additional 2 days. After
differentiation, cells were cultured for various times in DMEM
supplemented with insulin (5 µg/ml) and 10% FCS, with media changed
every other day. Mouse HC11 normal mammary epithelial cells (a gift
from Dr. Nancy Hynes, Friedrich Miescher Institute, Switzerland) were
routinely grown in RPMI 1640 supplemented with 10% FCS, 5 µg/ml
insulin, and 5 ng/ml epidermal growth factor (EGF, Collaborative Research, Bedford, MA). Cells seeded into six-well plates
(2 x 105 cells per well) were grown to
confluence (for
2 days) and then changed to various treatments for
an additional 3 days, with media changed every day. All cell culture
experiments were performed in duplicate or triplicate.
Statistical Analyses
Analyses were performed by ANOVA within StatView (SAS Institute,
Inc., Cary, NC), and means comparisons were performed by
Fishers Least Significant Difference test. Statistical
significance was considered at the 5% level.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Barbara K. Vonderhaar, Ph.D., Building 10, Room 5B47, National Institutes of Health, 10 Center Drive, Bethesda, Maryland 20892-1402. E-mail: bv10w{at}nih.gov
1 These authors were equal contributors. 
Received for publication November 15, 2000.
Accepted for publication February 15, 2001.
 |
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