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* Microvascular Research Laboratories, School of Veterinary Sciences, and
Henry Wellcome Laboratories for Integrative Neuroscience and Endocrinology, University of Bristol, Bristol, UK
1Correspondence: Microvascular Research Laboratories, Department of Physiology, Preclinical Veterinary School, University of Bristol, Southwell St., Bristol BS2 8EJ, UK. E-mail: D.O.B, dave.bates{at}bris.ac.uk; S.J.H., s.harper{at}bris.ac.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: angiogenesis • splicing • transgenic
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. It has been proposed that growth of new blood vessels occurs during this stage of mammary development (2)
and that the vascular growth is a requirement for alveolar development and function, particularly milk production (1)
. New blood vessel growth is regulated by a complex of different growth factors of the vascular endothelial growth factor (VEGF), angiopoietin, and ephrin families (3)
, which coordinately result in a novel functional vasculature in tissues where nutritional requirements increase (e.g., in the ovary, the endometrium, and in pathologies such as cancer) (4)
.
The VEGF family of proteins is required for many angiogenic mechanisms (4)
, but the role of VEGF in angiogenesis underlying alveolar differentiation still is unclear. VEGF is expressed in alveolar tissues, as determined by immunohistochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR), and VEGFRs (VEGF receptors) is present on the associated vasculature (5)
. VEGF is up-regulated by prolactin, the principal hormone driving mammary development in advance of lactation (6)
. The principal member of the VEGF family, VEGF-A, is a multifunctional cytokine (7)
, which has been shown to stimulate angiogenesis (8)
, increased vascular permeability (9)
to water (10)
and proteins (11)
, vasodilatation (12)
, and formation of fenestrations in endothelial cells (13)
. VEGF-A regulates developmental angiogenesis, and VEGF knockout mice are heterozygously lethal during early embryogenesis (embryonic day 8.5) (14)
. Recent experiments on mice with a conditional VEGF gene inactivation in alveolar myoepithelial cells have demonstrated that milk production is dependent on VEGF production by these myoepithelial cells (15)
.
Multiple isoforms of VEGF-A are generated by alternative splicing from a single gene (16
, 17)
. Alternate splicing of exons 6 and 7 provide varying heparin-binding affinity (7)
, and alternate splice acceptor sites in the terminal exon, exon 8, provide 2 different families of protein, which differ in their angiogenic activity (17
, 18)
. Proximal splicing in exon 8 results in the originally described VEGF isoforms, such as VEGF165, which are proangiogenic, increase vascular permeability, stimulate formation of fenestrae, and are vasodilators (4)
. Distal splicing, 66 bases downstream in exon 8, results in formation of a more recently described class of proteins, which includes VEGF165b, VEGF121b, and others (17
, 18)
. These isoforms, collectively known as VEGFxxxb, have been shown to inhibit VEGF165-mediated angiogenesis in rabbit cornea (19)
, rat mesentery (19)
, mouse dorsal skin chamber, and chicken chorioallantoic membrane (20)
. VEGFxxxb isoforms also have been shown to inhibit pathological angiogenesis in the mouse retina (21)
and in tumors (19
, 22)
. VEGF165b has been shown to bind to VEGFR2 and compete with VEGF165 for activation of cells expressing VEGFR2 (19
, 20)
, resulting in inhibition of a number of signal transduction pathways, including phosphorylation of MAPK, Akt, and p38MAPK (19
, 20)
. VEGFxxxb isoforms are the predominant isoforms in a number of normal tissues (22
; unpublished results), but appear to be down-regulated in angiogenic tissues, such as placenta (23)
, tumors (19)
, and diabetic vitreous (18
, 24)
.
To determine whether VEGF165b could inhibit VEGF-dependent angiogenesis in breast tissue and the physiological consequences of such VEGF inhibition, we generated a transgenic (TG) mouse line expressing VEGF165b under the control of the mouse mammary tumor virus promoter, which is up-regulated by prolactin and progesterone during the late stages of pregnancy in mammary epithelium.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Construction of pMMTV (mouse mammary tumor virus) VEGF165b
pcDNA3-VEGF165b was cloned as described previously (17)
. To generate a plasmid with MMTV-LTR (MMTV-long terminal repeat) upstream of the VEGF165b cDNA and poly(A) signal, pcDNA3-VEGF165b was digested with NruI to delete the CMV promoter, followed by creation of HindIII sites by ligation with a HindIII linker using a Rapid DNA Ligation kit (Roche Applied Science, Indianapolis, IN, USA). This product was recirculated, digested with KpnI, and then blunt ends were created with T4 DNA polymerase (Promega Corp., Madison, WI, USA). After HindIII digestion, a blunt end and a HindIII site were created at the 2 ends of the linear fragment. To get MMTV-VEGF165b-pA in the pcDNA3 backbone (without CMV), the MMTV-LTR from pMAMneo-Blue digested with HindIII and SmaI was inserted upstream of the VEGF165b cDNA in the linearized pcDNA3-VEGF165b with a Rapid DNA Ligation kit.
Transfection
MCF7 human breast cancer cells were cultured to 50% confluence. Equal amounts of pMMTV-VEGF165b and empty vector pMAM were transfected into MCF7 using the reagent Lipofectamine (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s suggestion.
Generation of TG mice
The DNA fragment of MMTV-VEGF165b-pA for microinjection was generated by HindIII and XmnI digestions of pMMTV-VEGF165b, and gel-purified using a QIAEX II DNA extraction kit (Qiagen, Valencia, CA, USA) before final purification with elutip minicolumns (Schleicher & Schuell Biosciences Inc., Keene, NH, USA) according to the manufacturer’s suggestion. Then, 5–10 ng/µl of purified DNA fragment was microinjected into the pronuclei of fertilized 1-cell stage embryos obtained from young C57BL6xCBA/CA mice. Successfully injected embryos were cultured overnight in M16 medium (Sigma-Aldrich Corp., St. Louis, MO, USA) at 37°C, 5% CO2 and transplanted into oviducts of pseudopregnant mice in C57BL6xCBA/CA background the next day. After pups were weaned, genomic DNA (gDNA) extracted from tail biopsies was screened for the existence of transgene via PCR and confirmed by Southern blotting.
PCR
PCR was performed with 1 pair of primers (forward primer sequence: 5'-TCA GCG CAG CTA CTG CCA TC-3' and reverse primer sequence: 5'-GTG CTG GCC TTG GTG AGG TT-3') giving rise to a PCR product of 208 bp to detect specifically the transgene. Another pair of primers (forward primer: 5'-ACG TCC TAA GCC AGT GAG TG-3' and reverse primer: 5'-CAG CCT TCT CAG CAT CAG TC-3') for mouse β-globin resulting in a band of 253 bp was also included in this amplification, serving as an internal control. Each reaction contained 2 µl of the 10x buffer; 0.2 mM dATP, dTTP, dCTP, and dGTP; 1.5 mM MgCl2; 500 nM forward and reverse primers; 0.5 U of Taq polymerase (ABgene); 0.5 µl gDNA; and water to 20 µl. PCR was initiated with 94°C for 4 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 62°C for 30 s, extension at 72°C for 30 s, and a final extension at 72°C for 10 min to finish.
Southern blotting
The 10–15 µg of tail genomic DNA was digested with SacI restriction enzyme and probed with an alkaline phosphatase-labeled fragment, the same as the DNA fragment used for microinjection. Probe preparation and transgene detection followed the manufacturer’s guideline of Gene Images Alkphos Direct Labeling and Detection System (Amersham, Little Chalfont, UK).
RT-PCR
Total RNA was isolated with Trizol (Invitrogen) extraction and DNase I (Invitrogen) digested according to manufacturer’s suggestion to prevent gDNA contamination. Then, 1 µg of DNase-treated RNA was reverse transcribed into cDNA with AMV reverse transcriptase using standard method as suggested by the manufacturer (Promega). Both cDNA and DNase-treated RNA were subjected to PCR with a forward primer 5'-ACA AGA TCC GCA GAC GTG TA-3' and a reverse primer 5'-ACA GAT GGC TGG CAA CTA GA-3' as above. A band at 199 bp indicated VEGF165b transgene expression.
Enzyme-linked immunosorbant assay (ELISA) of VEGFxxxb
Tissue protein lysate was prepared from both virgin and lactating mouse mammary tissue in radio-immuno-precipitation assay buffer. Protein concentration was determined with Bio-Rad assay (Bio-Rad Laboratories, Hercules, CA, USA), and the amount of VEGF165b in tissue was determined with ELISA, as described previously (22)
, with a specific detection antibody against VEGFxxxb isoforms.
Briefly, 0.08 µg of goat anti-VEGF polyclonal IgG (AF293-NA; R&D Systems Inc., Minneapolis, MN, USA) diluted in 1x PBS (pH 7.4) was adsorbed onto each well of a 96-well plate (Immulon 2HB; Thermo Life Sciences, Basingstoke, UK) overnight at room temperature. The plate was washed 3 times between each step with 1x PBS-Tween (0.05% v/v). After blocking with 100 µl of 5% w/v BSA in PBS for 1 h at 37°C, 100 µl of recombinant human VEGF165b (R&D Systems) diluted in 1% w/v BSA in PBS (ranging from 62.5 pg/ml to 4 ng/ml) or protein samples was added to each well. After incubation for 1 h at 37°C with shaking and 3 washes, 100 µl of mouse monoclonal anti-VEGFxxxb biotinylated IgG (clone 264610/1; R&D Systems) at 0.4 µg/ml was added to each well, and the plate was left for 1 h at 37°C with shaking. Then, 100 µl of streptavidin-HRP (R&D Systems) at 1:200 dilution in 1% w/v BSA in PBS was added; the plate was left at room temperature for 20 min, and 100 µl/well O-phenylenediamine dihydrochloride solution (Substrate reagent pack DY-999; R&D Systems) was added; and the plate was protected from light and incubated for 20 min at room temperature. The reaction was stopped with 50 µl/well 1 M H2SO4, and absorbance was read immediately in the Opsys MR 96-well plate reader (Dynex Technologies, Chantilly, VA, USA) at 492 nm, with control reading at 460 nm.
Whole-mount staining of mouse mammary tissue
Four inguinal mammary glands from both virgin and lactating mice were excised. The tissues were spread onto a glass microscope slide, fixed in Carnoy’s solution, and stained in carmine solution as described (25)
. Images were captured on a Nikon Eclipse microscope with a DCN100 camera (Nikon Instruments, Kingston-Upon-Thames, UK) under a x2 objective.
Hematoxylin and eosin (H&E) staining and determination of alveolar coverage area
Lactating-stage mammary tissues, after whole-mount staining, were embedded in paraffin wax, and 5-µm sections were subjected to H&E staining using standard procedure. Images were taken from 4 pairs of mouse mammary tissues, as above. Alveolar area was selected in Photoshop, and the covering area was counted with Image J software (National Institutes of Health, Bethesda, MD, USA).
Vasculature staining on mouse mammary tissues
Four inguinal mammary glands were fixed at 4°C overnight in 4% w/v paraformaldehyde dissolved in PBS. After a thorough wash in PBS, 6x 10 min each, fixed tissues were soaked in 30% w/v sucrose overnight before embedding in OCT (Vector Laboratories, Peterborough, UK). Then, 30-µm-thick sections were cut and dried thoroughly on Superfrost slides (Menzel Gläser, Braunschweig, Germany). Nonspecific binding was blocked with 5% w/v normal goat serum for 1 h, and Avidin/Biotin blocking reagent (Vector Laboratories) was applied for 30 min each on sections to block endogenous biotin. Vasculature staining was performed with incubation with 5 µg/ml biotinylated isolectin B4 (Molecular Probes Inc., Eugene, OR, USA) at 4°C overnight and washing in 0.5% v/v PBS-Triton 100 4 times each for 15 min, followed by 4 h incubation with 2 ng/ml TRITC-conjugated streptavidin (Vector Laboratories). After washing, sections were counterstained with 5 µg/ml Hoechst for 30 min and coverslipped with vectorshield (Vector Laboratories). Images from pairs of mammary tissues were analyzed with a Leica confocal microscope using a x40 oil objective. Microvessels stained in red were selected in Photoshop, and microvascular density (microvascular coverage area/total area %) was calculated in Image J.
VEGF staining on human breast tissues
Histologically normal, anonymized adult breast tissues from nonlactating women, formalin-fixed and embedded in paraffin, were obtained in accordance with the ethics committee approval from archives of tissues removed during surgery, from the Department of Histology, Southmead Hospital, Bristol, UK, and from the teaching archives of the Department of Physiology, University of Bristol. The 5-µm sections then were mounted onto gelatin/poly-L-lysine-coated glass slides. The sections then were dried onto the slides in a 37°C incubator overnight. Sections were dewaxed in Histoclear (RA Lamb, Eastbourne, UK) for 5 min and rehydrated through graded ethanol solutions (100, 90, and 70% v/v).
Microwave antigen retrieval was performed in 0.01 mM citric buffer, saturated sodium citrate pH buffer (pH 6.0), for 12 min at 95°C for VEGFxxxb staining and 7 min at 800 W followed by 9 min at 120 W for pan-VEGF staining. Sections were cooled to room temperature prior to being washed twice in deionized water for 5 min each time. Sections were incubated with freshly prepared 3% v/v hydrogen peroxide (BDH, Poole, UK) diluted in deionized water for 5 min, then washed twice for 5 min with PBS and blocked with 1.5% w/v normal goat serum (Vector Laboratories) in 10% w/v BSA for 30 min. The solution was washed twice with 0.05% v/v PBS-Tween at room temperature, then incubated with the primary antibody diluted in 1.5% w/v normal goat serum in 1x PBS. For VEGFxxxb, a monoclonal antiexon 8b specific antibody (MAB3045; R&D Systems) was used. This antibody has been fully characterized and has been shown to specifically recognize and be specific for VEGFxxxb isoforms using Western blotting and ELISA (18
, 24
, 26)
. For pan-VEGF staining, a monoclonal mouse anti-human VEGF antibody (sc-7269; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) was used. Tissue sections also were treated with a matched concentration of normal, affinity-purified mouse IgG (Sigma), used as a negative control. The sections were washed twice in 0.05% v/v PBS-Tween, for 5 min each time. The blocking step was repeated as before, followed by two 5 min washes in 0.05% v/v PBS-Tween. All sections, including the controls, then were incubated with biotinylated goat anti-mouse IgG (Vector Laboratories) diluted in 1.5% w/v normal goat serum for 1 h in a humid chamber at room temperature. Sections were washed twice with 0.05% v/v PBS-Tween, 5 min per wash, then incubated with a preprepared avidin-biotinylated enzyme complete kit (Vector Laboratories) for 45 min in a humid chamber at room temperature. Again, the sections were washed twice with 0.05% v/v PBS-Tween, 5 min each time, followed by incubation with 3,3'-diaminobenzidine substrate (Vector Laboratories) to yield a brown-colored product. The reaction was stopped by washing twice with deionized water for 5 min. Sections were counterstained with Mayer’s hematoxylin (BDH) for 5 min, then differentiated in water. Sections were dehydrated by passing through increasing concentrations of ethanol (70, 90, and 100% v/v) for at least 2 min each, cleared in xylene for at least 10 min, and permanently mounted in DPX mountant for histology. Staining was examined with a Nikon Eclipse E-400 microscope; images were captured using a DCN-100 digital imaging system (Nikon Instruments).
Statistical analysis
Fisher’s exact test was used in the pup survival experiments. Student’s 2-tailed t test and ANOVA were used for all others, with P < 0.05 as the level of significance.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A) and pan-VEGF (Fig. 1B
) staining localized to the cytoplasm of the myoepithelium of the alveoli and the epithelium of the interlobular ducts. VEGFxxxb and pan-VEGF expression also was seen in a variable portion of some of the plasma cells, fibroblasts, and lymphocytes of the loose connective tissue septa surrounding the lobule. No staining was observed with the nonspecific mouse IgG control (Fig. 1C
). At no point was pan-VEGF staining inconsistent with VEGFxxxb staining, but pan-VEGF staining was more extensive than VEGFxxxb staining, as areas that were not stained by antibodies to VEGFxxxb did stain for pan VEGF (Fig. 1)
. VEGF165b is the predominant isoform in the VEGFxxxb family in the normal human tissues we have so far investigated (unpublished results). Therefore, to determine whether VEGF165b expression may play a role in mammary gland physiology, we generated TG mice overexpressing VEGF165b during lactation.
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Generation of MMTV-VEGF165b mice
The VEGF165b cDNA was cloned into an MMTV expression vector (pMAM-Neoblue). To determine whether this vector resulted in expression of VEGF165b in mammary-derived cells in vitro, MCF7 breast cancer cells were transfected with the expression vector, and the supernatant was assayed for VEGF165b using an isoform-specific ELISA. Figure 2
A shows that transfection with this vector resulted in production of 1718.4 ± 249.6 pg/ml/48 h VEGF165b in the supernatant. Cells transfected with the empty vector or sham-transfected cells produced less than 25 pg/ml in 48 h (P<0.001). TG mice were generated by microinjection of vector fragment into fertilized 1-cell embryos from superovulated females, which were implanted into pseudopregnant recipient surrogate mothers. Viable offspring were screened by PCR of genomic DNA extracted from the tails using primers that detected the transgene specifically and a primer pair that detected the mouse β-globin gene (Materials and Methods). Figure 2B
shows that 2 independent TG founder mice were obtained (TG1 and TG2). The presence of the transgene was confirmed by Southern blotting. Figure 2C
shows that genomic DNA extracted from mouse tails and digested with EcoRI and hybridized to a VEGF probe showed fragments consistent with the MMTV-VEGF165b transgene in the 2 TG lines identified by PCR. The F0 generation was then crossed with a C57BL6 WT mouse and heterozygotes identified by PCR. The F1 generation of the TG1 strain (a male founder) was apparently normal and developed normally to adulthood. The TG2 strain, a female founder, produced no offspring. Figure 2D
shows WT and TG littermates of TG1 strain. The mice were active, alert, and appeared healthy.
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MMTV-VEGF165b TG mice express VEGF165b in the mammary gland
To determine whether the TG mice had significant expression of VEGF165b, tissues known to be permissive for MMTV expression in nonpregnant mice were examined. RNA was extracted from salivary, mammary, adrenal, and Harderian glands of adult mice and reverse transcribed. Figure 3
A shows that, while no band was seen in the absence of reverse transcription, expression was seen in all of these tissues in the TG mice when the RNA was reverse transcribed. WT mice did not show expression of human VEGF165b in the mammary tissue. Mammary tissue was assayed from mice sacrificed during pregnancy. Whereas transgene mRNA expression was weak in mammary tissue from virgin mice, expression increased during pregnancy and was clearly expressed immediately postpartum. No transgene expression was seen in mammary tissue of WT mice, even at parturition (Fig. 3B
). To quantify protein expression, VEGFxxxb (combined TG human VEGF165b and endogenous mouse VEGFxxxb) was measured in protein extracted from mammary tissues from TG and WT mice. Figure 3C
shows that VEGFxxxb increased from 2.27 ± 0.46 ng/mg in virgin TG mice to 4.69 ± 0.63 ng/mg postpartum (P<0.01 compared with virgin). In contrast, whereas WT virgin mice had similar expression levels to TG mice (2.26±0.19 ng/mg), expression was significantly down-regulated postpartum (0.475±0.04 ng/mg, P<0.001 compared with virgin). Interestingly, VEGF165 levels were the same in lactating tissue (11.1±2.5 ng/mg WT, 13.6±3.9 ng/mg TG) as in virgin mice (14.1±2.2 ng/mg WT, 16.1±1.9 TG, P>0.1 ANOVA).
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VEGF165b overexpression in mammary tissue results in pup lethality
Female F1 TG mice were mated with WT males. Surprisingly, most of the pups died within 2 days after birth. Of the 11 litters born to TG females, 8 of them died within 48 h of birth. This rate was significantly greater than in WT crosses (1 litter in 10 died), and greater than in litters of WT mothers mated with TG fathers (1 litter in 9 died, Fig. 4
A). To determine whether the deaths resulted from inherited defects in the pups, offspring from TG mothers were removed from the parents as soon as possible after birth and weighed, then transferred to foster mothers. There was no difference in pup weight between offspring of TG mothers when compared with WTs (Fig. 4B
). Moreover, pups from subsequent litters were effectively fostered in all cases onto WT mothers, suggesting a lactation-deficiency phenotype. To determine whether the offspring had any obvious internal phenotype, newborn mice were killed and the stomachs were examined at necropsy. Figure 4C
shows that the stomach was not distended and contained very little milk compared with WT pups, confirming that the offspring were not properly nourished.
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VEGF165b overexpression results in inhibition of mammary alveolar differentiation
To determine the effect of VEGF165b overexpression on ductal and alveolar development, mammary tissues were removed from WT and TG mice before pregnancy and at parturition. Figure 5
A shows WT ductal structure in a virgin mouse stained with carmine red. At parturition, the mammary epithelial structure was fully differentiated throughout the mammary gland, and extensive alveolar structures were seen (Fig. 5B
). In TG mice, ductal structures of virgin mice were normal (Fig. 5C
), but at parturition there was a reduction in alveolar structures when compared with WT (Fig. 5D
). Quantification of the area of alveolar coverage showed a significant reduction in coverage from 35 ± 3 to 17 ± 1.5% (Fig. 5E
; P<0.01, t test, n=4).
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VEGF165b overexpression results in inhibition of mammary angiogenesis
To determine the effect of VEGF165b expression on mammary angiogenesis, we investigated the vasculature of VEGF165b-expressing TG mice. Figure 6
A shows that the mammary fat pads of WT mice are erythematous when excised at parturition. In contrast, TG mouse mammary fat pads were pale with less blood apparent (Fig. 6B
). Sections of fat pad were stained with isolectin to visualize blood vessels. Figure 6C
shows that the WT tissue was highly vascularized (blood vessels in red), whereas the TG mice have many fewer blood vessels (Fig. 6D
). Quantitation of the blood vessel-covering area shows that the TG mice had a significantly lower vascular density than did WT animals (10.9±1.2% WT, 5.8±1.6 TG, Fig. 6E
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The final functional alveolar epithelial cells secrete milk into the glands. The milk is formed from the secretory epithelial cells and adipose tissue, which require an extensive vascular network of fenestrated endothelial cells (28)
. The results here indicate that expression of the proangiogenic isoforms of VEGF is required for production of milk. VEGF has been shown to be highly expressed during development of the mammary gland (5)
and is present in human milk in high concentrations (75 ng/ml in colostrums, 25 ng/ml after 3 days) (29)
. Up-regulation of VEGF at this time is accompanied by extensive angiogenesis and the formation of vascular honeycombs around the alveolar structures (30)
. Subsequent capillary remodeling has been shown to be modulated by intussusceptive growth (30)
, which is regulated by increased shear stress (31
, 32)
. This result also may be VEGF dependent because VEGF acts as a potent vasodilator. Recently, knockout of both VEGF families of isoforms has been shown to result in pup lethality, due to reduced milk production. This model used a mammary-specific Cre-Lox VEGF mouse in which exon 3 of VEGF, common to all isoforms, was excised under control of the 
5-keratin promoter, expressed in alveolar myoepithelial cells (15)
, which is a site of expression of both VEGF165b and VEGF165 (Fig. 1)
. This procedure resulted in knockout of VEGFxxxb as well as the VEGFxxx isoforms. We show here that VEGFxxxb isoforms are significantly down-regulated in mature mammary glands, which suggests that the reason for the milk deficit in the knockout was the result of the lack of VEGFxxx isoforms. This result—VEGF165b overexpression induces a similar phenotype—is consistent with that data—VEGF165-dependent angiogenesis is necessary for milk production—thus providing another example of VEGF165b demonstrating antiangiogenic activity, specifically in an endogenous, in vivo model of physiological angiogenesis. The outcome also provides further evidence for the concept that neovascularization is required for the expansion of tissue mass (33)
.
There are a number of mechanisms through which VEGF production may control the generation of milk. The differentiation of the alveolus is clearly inhibited by antiangiogenic VEGF isoform expression, possibly due to a lack of new vessel growth such that dividing alveolar epithelial cells are too far from a flowing vessel to continue to proliferate or differentiate. A lack of VEGF-mediated vasodilatation (12)
, hence shear stress, also could result in reduced intussusceptive growth, again reducing vascular density. The results shown here clearly indicate a paucity of alveolar structures, which would reduce milk production. Mammary capillaries during lactation have a high permeability (34)
. An additional mechanism that would further reduce milk production is the inhibition of vascular permeability induced by VEGF (10)
, resulting in reduced fluid filtration, hence fluid volumes (35)
, or an inhibition of the reduced pericyte coverage induced by VEGF (36)
, leading to altered microvascular phenotypes. VEGF165b, unlike VEGF165, does not chronically increase capillary hydraulic conductivity (37)
, but its effect on VEGF165 mediated permeability has not been measured. VEGF has been shown to induce fenestrated endothelial cells (13)
, and the fenestrated nature and permeability of the breast tissue may be inhibited by antiangiogenic VEGF isoforms.
In summary, we show here a reduction in vascular density and alveolar development in the VEGF165b TG mice, demonstrating for the first time a physiological role for VEGF165b, that of inhibition of physiological angiogenesis, and that alveolar development in the mammary gland is VEGF dependent.
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ACKNOWLEDGMENTS |
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Received for publication August 30, 2007. Accepted for publication October 25, 2007.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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