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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1AGo). 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. 1BGo).



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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.

 
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. 2Go). 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 7–10 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.

 
To determine the stromal expression of Flt-1 and Flk-1, CFP tissue was analyzed at various stages of development (Fig. 3Go). 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. 1Go. 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.

 
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. 4AGo), 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. 4BGo). 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. 4CGo). 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. 5Go, 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. 5CGo). Macrophages were also positive for VEGF (Fig. 5CGo). Within the lactating mammary gland, VEGF immunoreactivity localized primarily to the alveolar epithelium (Fig. 5DGo). 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 2–3 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.

 
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. 6Go), 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.

 
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. 7Go, 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. 7CGo and 8CGo). Within the lactating mammary gland, mRNA for both receptors localized basal to the alveolar epithelium (Figs. 7DGo and 8DGo) and became more punctate during established lactation (Figs. 7EGo and 8EGo). Expression of Flk-1 mRNA was also localized to brown adipose tissue in the lactating thoracic mammary gland (Fig. 7EGo). 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.

 
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. 9Go). 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. 10Go, A and B) concurrent with the induction of aP2 gene expression, a marker of adipocyte differentiation (Fig. 10CGo). 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. 10BGo). 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. 10DGo). 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.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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-{gamma}. 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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 1–1,2-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 6–8 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 21–23 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 2–3 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 4–8 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 manufacturer’s 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 Fisher’s 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. Back

Received for publication November 15, 2000. Accepted for publication February 15, 2001.


    REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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