Increased Pituitary Vascular Endothelial Growth Factor-A in Dopaminergic D2 Receptor Knockout Female Mice
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内分泌学杂志 2005年第7期
Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) (C.C., A.G., G.D.-T., A.B., D.B.-V., M.R.), Argentina; Instituto de Biología y Medicina Experimental (C.C., G.D.-T., A.B., D.B.-V.), 1428 Buenos Aires, Argentina; Instituto de Investigaciones en Ingeniería Genética y Biología Molecular (M.R.); University of Buenos Aires (M.R.), 1428 Buenos Aires, Argentina; and The Vollum Institute (M.J.L.), Oregon Health and Science University, Portland, Oregon 97239-3098
Address all correspondence and requests for reprints to: D. Becú-Villalobos, Instituto de Biología y Medicina Experimental, Consejo Nacional de Investigaciones Científicas y Técnicas, V. Obligado 2490, 1428 Buenos Aires, Argentina. E-mail: dbecu@dna.uba.ar.
Abstract
Vascular endothelial growth factor (VEGF)-A is an important angiogenic cytokine in cancer and pathological angiogenesis and has been related to the antiangiogenic activity of dopamine in endothelial cells. We investigated VEGF expression, localization, and function in pituitary hyperplasia of dopamine D2 receptor (D2R)-knockout female mice. Pituitaries from knockout mice showed increased protein and mRNA VEGF-A expression when compared with wild-type mice. In wild-type mice, prolonged treatment with the D2R antagonist, haloperidol, enhanced pituitary VEGF expression and prolactin release, suggesting that dopamine inhibits pituitary VEGF expression. VEGF expression was also increased in pituitary cells from knockout mice, even though these cells proliferated less in vitro when compared with wild-type cells, as determined by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium proliferation assay, proliferating cell nuclear antigen expression, and [3H]thymidine incorporation. In contrast to other animal models, estrogen did not increase pituitary VEGF protein and mRNA expression and lowered serum prolactin secretion in vivo and in vitro in both genotypes. VEGF (10 and 30 ng/ml) did not modify pituitary cell proliferation in either genotype and increased prolactin secretion in vitro in estrogen-pretreated cells of both genotypes. But conditioned media from D2R–/– cells enhanced human umbilical vein cell proliferation, and this effect could be partially inhibited by an anti-VEGF antiserum. Finally, using dual-labeling immunofluorescence and confocal laser microscopy, we found that in the hyperplastic pituitaries, VEGF-A was mostly present in follicle-stellate cells. In conclusion, pituitary VEGF expression is under dopaminergic control, and even though VEGF does not promote pituitary cellular proliferation in vitro, it may be critical for pituitary angiogenesis through paracrine actions in the D2R knockout female mice.
Introduction
THE DOPAMINE D2 receptor (D2R) is the predominant dopamine receptor subtype in the anterior pituitary and mediates dopamine’s inhibitory actions on lactotrophs (1, 2). Recently the importance of D2R in maintaining normal lactotroph function has been clearly demonstrated. Mice lacking D2R generated by homologous recombination in embryonic stem cells display chronic hyperprolactinemia and lactotroph hyperplasia (3, 4). Pituitary adenoma growth, as with all tumors, depends on adequate vascularization. In rats it has been shown that estrogen-induced prolactin-secreting pituitary tumors are highly angiogenic (5), and furthermore tumor growth can be blocked by antiangiogenic agents (6, 7). In D2R knockout mice, prominent vascular channels have been described in the hyperplastic pituitaries as well as extravasated red blood cells or peliosis (8).
Cytokines and growth factors are important modulators of angiogenesis. One of these factors is the vascular endothelial growth factor-A, (VEGF) a dimeric N-glycoprotein of relative molecular mass of 43–46 kDa, formerly described as a permeability factor. VEGF is a potent mitogen for micro- and macrovascular endothelial cells derived from arteries, veins, and lymphatics but not for other cell types (9). Enhanced VEGF expression has been associated with several human vascular tumors, including brain, colon, gastrointestinal tract, ovary, breast, and others (9). Furthermore, this glycoprotein is also abundantly expressed and/or secreted by most animal tumors. Moreover, studies on tumor angiogenesis in nude mice indicate that VEGF expression is critical for effective tumorigenesis and tumor angiogenesis (10, 11).
VEGF has been detected in all types of human pituitary adenomas, primarily in those of somatotrophic or corticotrophic type (12, 13). Furthermore, increased concentrations of VEGF and the VEGF receptor (VEGF-R)2 (KDR, or Flk-1) have previously been reported in rat pituitary tumors (14, 15). The VEGF system plays a crucial role not only in the regulation of tumor angiogenesis during the development of estrogen-induced prolactin secreting pituitary tumors (14) but also in the formation of pituitary portal vessels during fetal life and in the maintenance of their differentiated state in adult animals (16).
The participation of VEGF in pituitary hyperplasia in D2R knockout mice has not been described to date. Even though these animals have low serum estrogen, the loss of the dopamine inhibitory control on lactotrophs may result in a permissive environment for stimulatory factors to function unopposed, leading to lactotroph proliferation, angiogenesis, and tumor development.
Consistent with the presence of activator protein-1 and -2 sites in the VEGF gene promoter, phorbol esters and forskolin that activate adenylate cyclase induce VEGF mRNA expression (17). Besides, several reports have shown that increases in cAMP production stimulate VEGF gene expression (18) (19). In the pituitary of the D2R knockout mice, the lack of action of dopamine on its receptor prevents physiological adenylate cyclase inhibition. Therefore, it was of interest to study the expression levels of VEGF in pituitary cells from D2R knockout mice. Furthermore, in endothelial cells it has been described that dopamine has antiangiogenic activity mediated through the D2R, inhibiting malignant tumors as well as the vascular permeabilizing and angiogenic activities of VEGF (20). Another link of the dopaminergic function and VEGF expression has been reported in two outbred lines of Wistar rats, which present high and low dopaminergic reactivity, respectively. VEGF expression was reduced in the first group, which was more resistant to tumor implantation, and developed significantly fewer lung metastases (21). Finally, in gastric cancer tissues, a low nontoxic dose of dopamine significantly retarded tumor angiogenesis by inhibiting VEGF-R2 phosphorylation within the tumor endothelial cells expressing D2Rs (22).
Therefore, in view of the angiogenic properties of VEGF as well as its relation to dopamine activity in other tissues, we decided to analyze its expression, localization, and regulation by estrogen in pituitary hyperplasia in the D2R knockout female mouse. In addition, we studied the effects of VEGF on prolactin secretion and pituitary cell proliferation in wild-type and D2R knockout female mice.
Materials and Methods
Animals
D2 dopamine receptor knockout mice (official strain designation B6; 129S2-Drd2tm1low by the Induced Mutant Resource at The Jackson Laboratory, Bar Harbor, ME), generated by targeted mutagenesis of the D2R gene in embryonic stem cells (3, 8) were used. The original F2 hybrid strain (129S2/Sv X C57BL/6J) containing the mutated D2R allele was backcrossed for eight generations to wild-type C57BL/6J mice. Mutant and wild-type mice were generally the product of heterozygote crossings, and in all cases sibling controls were used. Female mice were housed in groups of four or five with mixed genotypes in an air-conditioned room with lights on at 0700 h and off at 1900 h. They had free access to laboratory chow and tap water. Wild-type, heterozygous and knockout mice were identified by PCR of genomic DNA, as previously described (23). Animals were used at 8–10 months, and pituitaries from knockout females were hyperplastic at this moment. All experimental procedures were reviewed and approved by the Institutional Animal Care and Use Committee of the Instituto de Biología y Medicina Experimental, Buenos Aires (Division of Animal Welfare, Office for Protection of Research Risks, National Institutes of Health, A#5072–01).
Drugs
Unless specified, all chemicals were purchased from Sigma (St. Louis, MO).
In vivo experiments
Wild-type 8-month-old female mice were divided in groups and treated with castor oil (controls) or haloperidol-decanoate (HALOPIDOL; Janssen-Cilag, Beerse, Belgium), a long-acting D2 antagonist, in a dose of 5 mg/kg, sc, for 3 wk, one injection per week, or 1.2 mg/kg ip for 7 d, one injection per day.
Two other groups of wild-type female mice were injected either with saline solution (controls) or cabergoline (0.5 mg/kg sc; Beta Laboratories, Buenos Aires, Argentina) for 3 wk, two injections per week. Other groups were treated with estradiol-valerate (Progynon Depot; Schering, Buenos Aires, Argentina), 0.2 mg/kg sc or castor oil for controls, 72 and 24 h before sampling. After treatment blood was collected by decapitation. Sera were kept at –20 C until RIAs were performed. Pituitaries were excised as described below for Western blot analysis.
Cell dispersion and culture
Anterior pituitaries from 8- to 10-month-old female wild-type and knockout mice were weighed and dissociated into single cells. Anterior pituitaries were placed in chambers containing freshly prepared Krebs-Ringer bicarbonate buffer without Ca2+ or Mg2+. Buffer contained 14 mM glucose, 1% BSA, 2% MEM amino acids, 1% MEM vitamins (Life Technologies, Inc., Buenos Aires, Argentina), and 2 mM glutamine and was previously gassed during 15 min with 95% O2-5% CO2 and adjusted to pH 7.35–7.40. Buffer was filtered through a 0.45-μm pore diameter membrane (Nalgene, Rochester, NY). Pituitaries were washed three times with Krebs-Ringer bicarbonate buffer and then cut into 1-mm pieces. Fragments obtained were washed and incubated in the same buffer containing 0.5% trypsin for 30 min at 37 C in 95% O2-5% CO2, followed by 2 additional min with 50 μl deoxyribonuclease I (1 mg/ml; Worthington Biochemical Corp., Lakewood, NJ). Digestion was ended by adding 1 mg/ml lima bean trypsin inhibitor. Fragments were disassociated to single cells by gentle trituration through Pasteur pipettes. The resulting suspension was filtered through a nylon gauze (160 μm pore size) and centrifuged 10 min at 1000 x g. Before centrifugation, an aliquot of cellular suspension was taken to quantify pituitary cell yield, using a Neubauer chamber. Viability of cells, determined by Trypan Blue exclusion, was always greater than 90%. Cells (35,000, 250,000, or 375,000 cells/well, depending on the type of assay) were cultured for 5 d in DMEM, 10% horse serum, 2.5% fetal bovine serum (FBS) with our without 10–8 M 17?-estradiol. Cells were then washed and stimulated with 10 or 30 ng/ml recombinant human VEGF-A for 48 h in DMEM 0.5% BSA medium, without serum (with our without estradiol). Cell culture was performed as previously described (24).
Conditioned media (CM) were obtained after culturing 250,000 or 375,000 cells in 24-well plaques for 5 d in the presence of 2.5% FBS (Life Technologies), followed by 2 d without serum, as above described. CM (800 μl) were collected and cells were counted. For RIA assays CM was concentrated (1:10) using a lyophilizer.
RIAs
Prolactin was measured by RIA using a kit provided by the National Institute of Diabetes and Digestive and Kidney Diseases [NIDDK; Dr. A. F. Parlow, National Hormone and Pituitary Program (NHPP), Torrance, CA]. Assays were performed using 10 μl serum in duplicate or the adequate quantity of diluted medium from cultured cells. Results are expressed in terms of mouse prolactin RP3. Intra- and interassay coefficients of variation were 7.2 and 12.8%, respectively.
VEGF
A RIA was developed according to the method described by Anthony et al. (25). Recombinant human VEGF165, the main form of VEGF-A, was used as standard and tracer. VEGF was labeled with 125I (NEN Life Science Products, Inc., Boston, MA) using a modified chloramine T method for iodination. Briefly, 2 μg VEGF was iodinated with 0.7 mCi 125I in 0.5 M phosphate buffer in the presence of 16 μg chloramine T for 45 sec. Reaction was stopped with sodium metabisulfite and the reaction transferred to a Biogel P10 column (Bio-Rad Laboratories, Hercules, CA) previously blocked with 2% BSA in 0.01 M phosphate buffer and 50 mM EDTA. RIA incubation mixtures consisted of 200-μl aliquots of standard (36 to 0.28 ng/tube) or samples in assay buffer [200 mg/liter protamine sulfate, 4.14 g/liter sodium phosphate (monobasic), 0.05% Tween 20 (Promega, Madison, WI), 0.02% sodium azide, and 0.01 M EDTA, 200 μg/ml heparin (Gibco, Buenos Aires, Argentina) (pH 7.5)] containing 100 μl of radioactive tracer 10,000 cpm and 100 μl of rabbit polyclonal VEGF antiserum (1:5,000, sc-507; Santa Cruz Biotechnology, Santa Cruz, CA). After overnight incubation at 4 C, 0.5 ml of 0.01 M PBS containing 5% polyethylene glycol 6000 (Merck-Schuchardt, Hohenbrunn, Germany), 1% (vol/vol) sheep antirabbit -globulin, and 0.05% normal rabbit serum were added to the tubes. After a 2-h incubation at room temperature and an hour at 4 C, tubes were centrifuged at 2000 x g for 30 min at 4 C. The supernatant was aspirated and the radioactivity in the remaining pellet was measured. Assay sensitivity was 0.6 ng/tube. The intraassay coefficient of variation was 10%, and parallelism was obtained between diluted conditioned media from mouse pituitaries and standard human curve. No cross-reactivity was found with basic fibroblast growth factor (FGF) or IGF-I.
Western blot
Anterior pituitaries were homogenized in 80 μl ice-cold buffer containing 60 mM Tris-HCl, 1 mM EDTA (pH 6.8), and a mix of proteases inhibitors in a handheld microtissue homogenizer. The homogenate was then centrifuged at 800 x g for 5 min at 4 C. An aliquot of supernatant was taken to quantify proteins by the Lowry method. Thirty micrograms of proteins in 10 μl of buffer and 60 mM Tris HCl (pH 6.8) were mixed with 10 μl 2 x sample buffer [60 mM Tris-HCl, 4% sodium dodecyl sulfate, 20% glycerol, 0.02% bromophenol blue, and 50 mM dithiothreitol (pH 6.8)]. Samples were sonicated during 20 sec and heated 5 min at 95 C and subjected to 12% SDS-PAGE. The gel was then blotted onto a nitrocellulose membrane (Bio-Rad) and probed with the corresponding primary antibody followed by a secondary antibody conjugated with horseradish peroxidase. Polyclonal rabbit VEGF antibody (1:1000, sc-507; Santa Cruz Biotechnology) was used. This antibody recognizes the 189-, 165-, and 121-amino acid splice variants of VEGF-A. The visible band corresponded to VEGF165, and in some cases a doublet of this band was observed. Proliferating cell nuclear antigen [PCNA (FL-261): sc-7907] and estrogen receptor- [ER (MC-20), sc-542] antibodies were purchased from Santa Cruz Biotechnologies. Monoclonal mouse actin antibody (Ab-1) was purchased from Lab Vision Co. (Fremont, CA). Immunoreactive proteins were detected by enhanced chemiluminescence (Amersham, Buenos Aires, Argentina). For repeated immunoblotting, membranes were incubated in stripping buffer [62.5 mM Tris, 2% sodium dodecyl sulfate, and 100 mM mercaptoethanol (pH 6.7)] for 40 min at 50 C and reprobed. Band intensities were quantified using the ImageQuant software.
Preparation of pituitary RNA
Total RNA was isolated from anterior pituitaries using TRIzol reagent (Gibco). Each gland was homogenized in 100 μl TRIzol, sonicated for 10 sec, and incubated at room temperature for 5 min. Chloroform (20 μl) was added, samples were shaken vigorously, and after 5 min of incubation at room temperature, they were centrifuged at 12,000 x g for 15 min at 4 C. Isopropanol (50 μl) was added to the supernatant to precipitate the RNA. After a 10-min incubation at room temperature, samples were centrifuged at 12,000 x g for 10 min at 4 C, supernatants discarded, and their pellets washed with 100 μl of 70% ethanol. The resulting precipitates were resuspended in 5 μl diethylpyrocarbonate-treated water. RNA was quantified by UV spectrophotometry and its integrity checked by gel electrophoresis.
Semiquantitative RT-PCR
Total RNA (1.5 μg) was reverse transcribed in a reaction mixture containing 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol, 1 mM deoxynucleotide triphosphates, 8 U Rnase inhibitor (Promega), 1 μg of random hexamers (Biodynamics SRL, Buenos Aires, Argentina), and 200 U Moloney murine leukemia virus transcriptase (Invitrogen Life Technologies, Buenos Aires, Argentina) in a final volume of 20 μl. After incubation at 37 C for 60 min, samples were heated for 10 min at 70 C to inactivate the transcriptase. The product was amplified with VEGF and glycerol-3-phosphate dehydrogenase (G3PDH) sense and antisense primers in a reaction mixture (30 μl) containing 20 mM Tris-HCl (pH 8.4), 50 mM KCl, MgCl2 (see Table 1), 0.2 mM deoxynucleotide triphosphates, 0.5 μM of each primer, and Taq DNA polymerase (Invitrogen Life Technologies), using an Eppendorf thermal cycler.
TABLE 1. Description of primers used and PCR conditions
In Table 1, primer sequences and conditions of PCR amplification are detailed. For VEGF, primers were chosen to detect all splice variants of VEGF-A because they were directed against the common area of the cDNA (26). Common steps were a hot start step of 3 min at 95 C, followed by n cycles of denaturation at 94 C for 60 sec, annealing for 60 sec, and extension at 72 C for 50 sec, with a final elongation step of 5 min at 72 C.
Preliminary experiments using various RNA concentrations and cycle numbers confirmed that these PCRs were performed within the linear phase of the PCR amplification reaction. Ten microliters of amplified mixture were mixed with 1 μl of sample buffer (25% bromophenol blue, 30% glycerol) and analyzed by 1.8% agarose gel electrophoresis. The amplified DNA bands were detected by ethidium bromide staining. Densitometric analysis was conducted using the Scion Image software and intensity values of VEGF PCR products were normalized to the corresponding G3PDH products.
DNA synthesis in pituitary cells in culture
Culture procedure was the same as described above. [3H]thymidine (0.2 μCi/well, 87.7 Ci/mmol, NEN Life Science Products) was added to cultures. After 24 h. of incubation, medium was discarded and the cells were removed and lysed by treatment with 0.05% trypsin and 0.02% EDTA in deionized water. The reaction was stopped 20 min later by filtering under vacuum through GF/C filters (Whatman, Middlesex, UK) using the Cell Harvester 8 (Nunc, Glastrup, Denmark). After five washes with deionized water, the filters were placed in plastic vials with 3 ml scintillation solution and radioactivity counted in a Beckman counter. Each experiment was repeated six times.
Cell proliferation assay
Proliferation of anterior pituitary cells was also colorimetrically determined at 490 nm using a commercial proliferation assay kit (CellTiter 96 AQueous nonradioactive cell proliferation assay; Promega). After incubation with various concentrations of VEGF-A for 48 h, cells in a 96-well plate were incubated with 333 mg/liter 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) and 25 μM phenazine methosulfate solution for 0.5, 1, and 2 h at 37 C in a humidified, 50 ml/liter CO2 atmosphere. The absorbance of soluble formazan produced by cellular reduction of MTS was measured at 490 nm using an ELISA reader (Sensident scan; Merck). In each experiment four to six mice of each genotype were used, experiments were repeated four times, and each had quadruplicate samples.
To evaluate the time curve of pituitary cell proliferation comparatively between genotypes, cells were dispersed on d 1 and plated in quadruplicate in eight individual 96-well plates. Proliferation was assayed at time 0, 5 h, and 1, 2, 3, 4, 5, and 7 d as described above. In one set of experiments on d 5, medium was refreshed, and cells were cultured for 4 additional days in medium with serum. Experiment was repeated three times.
Human umbilical cord vein endothelial cell (HUVEC) culture and proliferation assay
Endothelial cells were isolated from HUVECs by enzymatic digestion with collagenase as previously described (27). HUVECs were cultured in T75 flasks in M199 supplemented with 20% FBS, growth factors, and 50 μg/ml gentamicin and were maintained at 37 C in a fully humidified atmosphere of 5% CO2 in the air. The culture medium was changed every 72 h, and HUVEC confluent cultures were washed twice with PBS, released with 0.05% (wt/vol) trypsin and 5 mM EDTA and subcultured. Cell proliferation studies were carried out using endothelial cells at passages 6–9.
The proliferation of HUVECs was measured by [3H]thymidine incorporation. HUVECs were harvested with trypsin/EDTA and suspended in M199 (supplemented with 20% FBS and 50 μg/ml gentamicin) at a density of 25,000 cells/ml and then seeded into a 96-well plate (100 μl per well: 2500 cells/well) and incubated for 2 h for attachment. Then 50 μl of the same medium with murine VEGF-A (final concentration 10 ng/ml), basic FGF (final concentration 2 ng/ml), epidermal growth factor (EGF, 1 ng/ml), or various CM were added alone or with 2.5 μg/ml polyclonal antibody against VEGF-A. They were incubated for 24 h before adding 5 μCi/ml [3H]thymidine. After 48 h incubation, the assay was ended by adding 50 μl guanidine-HCl, and the cells were lysed by a freezing-defrost cycle. The DNA was harvested in Whatman GF/C filters by using an 8-well harvester (Cell Harvester 8; Nunc), and 1 ml scintillation solution (OptiPhase Hifase 3) was added. The [3H]thymidine incorporation was measured by using a liquid scintillation counter.
Double-labeling immunofluorescence and confocal laser microscopy
Double-labeling immunofluorescence was applied to specifically identify the cell type(s) expressing VEGF-A. Double immunostaining was performed on paraffin-embedded sections of D2R knockout mice of the 129S6 and C57BL/6 congenic strains. We combined chicken anti-VEGF antibody with rabbit polyclonal antibodies against pituitary hormones or S-100 protein, a marker of folliculostellate cells. Specifics of the various antisera employed were as follows: chicken polyclonal to VEGF (dilution 1:100, ab 14078; Abcam, Cambridge MA), rabbit antisera directed against mouse prolactin (dilution 1:500; NHPP, NIDDK-AFP-107120402), mouse GH (dilution 1:1000, NHPP, NIDDK-AFP-5672099), rat TSH (?-TSH) (dilution 1:500; NHPP, NIDDK-AFP-1274789), rat LH (?-LH) (dilution 1:1,200; NHPP, NIDDK-AFP-571292393), and bovine S-100 protein (1:200, Ab2; Lab Vision). After rinsing in PBS, the double-stained sections were incubated at room temperature for 2 h with fluorescein isothiocyanate goat antirabbit IgG (dilution 1:100; Zymed Laboratories Inc., San Francisco, CA) and Texas red-X-conjugated goat antichicken IgY (dilution 1:100, Sc-2994; Santa Cruz Biotechnologies). After rinsing in PBS, the sections were mounted in Vectashield (Vector Laboratories, Burlingame, CA) to prevent fading of the immunofluorescence reaction. Sections were examined on a C1 Plan Apo x60/1.4 oil confocal laser-scanning system (Nikon, Tokyo, Japan). The excitation wavelength was 488 nm for fluorescein isothiocyanate and 543 nm for Texas red-X-induced fluorescence. Specificity studies were carried out by omitting primary antisera or preabsorbing the primary antisera with homologous antigen excess; all showed the absence of the fluorescent signal.
Statistical analyses
Results are expressed as means ± SE. VEGF expression, ER expression, and prolactin release in vivo and in vitro were analyzed by two-way ANOVA for independent measures for the effects of genotype and estrogen treatment. PCNA expression in vivo, VEGF-A release in vitro, PCNA expression in vitro, 3(H)-thymidine uptake, MTS assay, and haloperidol and cabergoline effects were analyzed by one-way ANOVA. Effect of CM on proliferation of HUVECs, and proliferation time curve were analyzed by two-way ANOVA for repeated measures. In all cases, if F of interaction was found significant, individual means were compared by Tukey’s honest significant difference or Fisher’s protected least significant difference tests; if it was not significant, groups of means were analyzed by the same tests. P < 0.05 was considered significant.
Results
When compared with female wild-type mice, pituitaries from female D2R knockout mice had increased VEGF concentration (normalized to actin content, Fig. 1A; P = 0.0036) as well as greater VEGF mRNA expression (normalized to the housekeeping gene G3PDH, Fig. 1B; P = 0.043). Enhanced PCNA concentration was also found in pituitaries from female knockout mice (Fig. 1C; P = 0.026) in concordance with increased pituitary weight as previously described (23) (pituitary weight in milligrams ± SE was 7.03 ± 1.39 and 2.51 ± 0.34 for knockout and wild-type mice, respectively; P = 0.011).
FIG. 1. A, Comparative pituitary VEGF-A content (evaluated by Western blot) in wild-type (+/+, open bars) and D2R knockout (–/–, filled bars) female mice. For each sample, arbitrary units of band intensities for VEGF-A were divided by band intensities for the respective actin and compared with those of wild-type females (considered 100%) in each series of experiments. *, P < 0.05 vs. +/+; n = 18 and 15, respectively. For this and following figures, results shown are means ± SE. B, Densitometric analysis of pituitary VEGF-A RT-PCR products in wild-type (open bars) and D2R knockout (filled bars) female mice. For each sample, intensity units of the VEGF band were normalized to those of the respective G3PDH band. *, P < 0.05 vs. +/+; n = 5 and 6. C, Pituitary PCNA content (evaluated by Western blot) in wild-type (+/+, open bars) and D2R knockout (–/–; filled bars) female mice. For each sample, arbitrary units of band intensities for PCNA were divided by band intensities for the respective actin and compared with those of wild-type females (considered 100%) in each series of experiments. *, P < 0.05 vs. +/+; n = 9 and 11, respectively. Below each graph representative bands are depicted.
A group of female wild-type mice was treated for 1 or 3 wk with a long-acting D2R antagonist, haloperidol-decanoate. We found that the longer treatment evoked a significant increase in pituitary VEGF concentration (Fig. 2A; P = 0.0029). As expected, prolactin levels also rose in this group (Fig. 2C; P = 0.0064). On the other hand, a 3-wk treatment with the D2R agonist cabergoline lowered prolactin levels in female wild-type mice (Fig. 2D; P = 0.0012) but did not significantly decrease pituitary VEGF expression (Fig. 2B; P = 0.57).
FIG. 2. Left panels, Effect of in vivo haloperidol treatment in wild-type females on pituitary VEGF-A content, normalized to the respective actin content of the sample (A) and serum prolactin levels (nanograms per milliliter) (C). Oil-injected females (oil) were considered as 100% in A. hal1, One week of haloperidol decanoate (see Material and Methods); hal3, 3 wk of haloperidol deca-noate treatment (n = 10, 7, and 6, respectively). Right panels, Effect of prolonged in vivo cabergoline (CB) treatment in wild-type females on pituitary VEGF-A content (B) and serum prolactin levels (nanograms per milliliter) (D) (n = 6 for each group). *, P < 0.05 vs. control (SAL). Below A and C are representative bands.
We next determined VEGF cellular expression and secretion, prolactin secretion, and basal cell proliferation in cultured pituitary cells from wild-type and knockout female mice. VEGF concentration in cells, measured by Western blot and normalized to actin content, was also increased in pituitary cells from knockout females (Fig. 3A; P = 0.018). Furthermore, in conditioned media from cells from knockout mice, higher VEGF and prolactin, measured by RIA, were found (Fig. 3, B and C; P = 0.043 and 0.0099, respectively).
FIG. 3. A, VEGF-A content (Western blot) in pituitary cells cultured in vitro (n = 4). B,VEGF-A release (nanograms per milliliter, RIA) from pituitary cells cultured in vitro (n = 6). C, Prolactin release in vitro (n = 4). *, P < 0.05 vs. respective wild type.
However, the rate of cell proliferation in this group was lower when compared with wild-type mice as determined using the MTS proliferation assay, PCNA expression by Western blot, and [3H]thymidine incorporation (Fig. 4, A–C; P = 0.000155, 0.039, and < 0.00001 for the three assays, respectively). Using identical conditions to the experiments described above (cells were cultured for 5 d with serum and serum starved for 2 additional days), we performed a proliferation time curve with cells from both genotypes and found that during the first 5 h, cells from wild-type pituitaries had a higher proliferation rate in comparison with their knockout counterparts; thereafter cells from both genotypes exhibited a parallel proliferation curve, with higher number of cells (as assayed by the MTS test) in wild-types (Fig. 4D). In a new set of experiments, at 5 d medium was changed and cells were cultured for 4 additional days in the presence of serum; differences in MTS assay were maintained (OD ± SE for wild-type and knockout: 0.0169 ± 0.001 and 0.068 ± 0.011, respectively; P < 0.01).
FIG. 4. A, MTS proliferation assay. Results are expressed as OD at 490 nm (n = 4). B, PCNA content in cultured pituitary cells (n = 5). C, [3H]thymidine incorporation in cultured pituitary cells (n = 9). D, Proliferation time curve (MTS assay; 35,000 original cells per well; n = 4). *, P < 0.05 vs. respective wild type.
As described, whole CM was first analyzed for the presence of VEGF-A by RIA. The results showed that VEGF was present in CM from both genotypes and that the concentration was higher in CM from D2R–/– cells (Fig. 3B). We next tested the effect of CM on DNA synthesis in endothelial cells by the measurement of [3H]thymidine incorporation into DNA in confluent quiescent HUVECs in the presence and absence of whole CM from anterior pituitary cells from both genotypes. The addition of CM from both genotypes to quiescent cells led to a significant increase in DNA synthesis in HUVECs, but the effect was significantly higher in CM from D2R–/– cells (P = 0.0024 vs. wild-type cells, Fig. 5A). There was a significant interaction for the effects of anti-VEGF pretreatment and group F(2, 9) = 4.35, P = 0.048. The proliferating effect was decreased in the presence of anti-VEGF in CM from D2R–/– (P = 0.038) and not from wild-type cells (P = 0.23). As an internal control of proliferation, we determined that addition of VEGF (10 ng/ml), EGF (1 ng/ml), and basic FGF (2 ng/ml) markedly increased [3H]thymidine incorporation, and only the effect of VEGF was blocked by anti-VEGF pretreatment (Fig. 5B).
FIG. 5. Effect of CM obtained from pituitary cells from knockout (KO) or wild-type (WT) female mice on HUVEC proliferation. A, HUVECs were grown to confluence and then exposed to CM or serum-free DMEM in presence or absence (buffer) of anti-VEGF (2.5 μg/ml). Proliferation was estimated as the incorporation of [3H]thymidine into DNA (counts per minute). , P = < 0.05 vs. WT. *, P < 0.05 vs. respective buffer (n = 4). B, Internal controls of HUVEC proliferation, murine VEGF-A (10 ng/ml), EGF (1 ng/ml), FGF-2 (2 ng/ml), n = 3.
We tested whether estrogen treatment in vivo could increase pituitary VEGF concentration in both genotypes. We found that estrogen did not increase VEGF concentration (protein or mRNA) in D2R knockout mice (Fig. 6, A and B; effect of estrogen on VEGF protein or mRNA was P = 0.78 and 0.27, respectively). This was not due to lack of pituitary ER, which was increased in knockout mice and could be down-regulated by estrogen treatment (Fig. 6C; P = 0.044 and 0.00070 for the effects of genotype and estrogen treatment; interaction P = 0.21).
FIG. 6. Effect of estradiol valerate (estrogen) treatment in vivo (0.2 mg/kg sc or castor oil for controls 72 and 24 h before sampling) on pituitary VEGF-A content evaluated by Western blot and normalized to the respective actin content of the sample (A); pituitary VEGF mRNA evaluated by RT-PCR and normalized to respective G3PDH mRNA (B); pituitary ER by Western blot normalized to actin content (C); and serum prolactin (nanograms per milliliter) (D). E, Effect of 10–8 M estradiol in vitro on prolactin release in cultured pituitary cells from donor wild-type and knockout females. *, P < 0.05 vs. respective oil-treated control. , P < 0.05 vs. treatment-matched wild-type mice. N = 18, 14, 15, and 19 (A); 5, 4, 6, 6 (B); 5, 7, 6, and 7 (C); 29, 25, 23, and 22 (D); 4 for each group (E).
VEGF protein and mRNA concentration were not increased either by estrogen in wild-type C57BL/6 mice, in contrast to other experimental models (14, 28). But also in contrast to other data reported, estrogen lowered serum prolactin release in vivo and prolactin secretion in vitro in both genotypes [Fig. 6, D and E; P interaction (genotype x estrogen) = 0.12 and 0.96; P effect estrogen = 0.0015 and 0.00015 for in vivo and in vitro, respectively].
Because pituitary VEGF was increased in D2R knockout females, we evaluated whether VEGF could affect pituitary cellular proliferation and/or prolactin secretion in both genotypes in the presence or absence of estrogen in vitro. VEGF (10 and 30 ng/ml) did not modify cell proliferation in either genotype, treated or not with estrogen (Fig. 7, A and B; P = 0.74 and 0.43 for the effect of drug in wild-type and D2R knockout, respectively) in contrast to its effect on endothelial cells. Similar results were obtained with PCNA/actin expression in buffer-pretreated cells (data not shown). Even though VEGF did not affect prolactin secretion in buffer-pretreated cells of either genotype, it significantly increased prolactin release in both genotypes when estrogen was added to the medium (Fig. 8, A and B; P = 0.039 and 0.027, VEGF 10 ng/ml vs. buffer for wild-type and knockout, respectively).
FIG. 7. MTS proliferation assay in cultured pituitary cells from wild-type (A) and knockout mice (B). Cells were pretreated with buffer (empty bars) or 10–8 M estradiol (5 d, hatched bars) and then stimulated with buffer (ctrol) or VEGF (10 or 30 ng/ml, VEGF10 and VEGF30) in buffer with or without estradiol for 48 h. Results are expressed as OD at 490 nm (n = 4 for each group).
FIG. 8. Prolactin release in cultured pituitary cells from wild-type (A) and knockout mice (B). Cells were pretreated with buffer (empty bars) or 10–8 M estradiol (5 d, hatched bars) and then stimulated with buffer (Ctrl) or VEGF (10 or 30 ng/ml, VEGF10 and VEGF30) in buffer with or without estradiol for 48 h. Results are expressed as percent release with respect to buffer-treated cells (n = 4 for each group). P < 0.05 vs. respective genotype- and pretreatment-matched control (Ctrl).
Double-labeling immunofluorescence and confocal laser microscopy was applied to specifically identify the cell type(s) expressing VEGF-A in pituitaries from D2R knockout mice. As shown in Fig. 9, A–E, VEGF-A strongly colocalized with the S-100 protein and not with prolactin, TSH, LH, or GH. Besides, we found histologic evidence of peliosis (Fig. 9E).
FIG. 9. Double-label immunofluorescent staining combined with confocal laser microscopy in the pituitary of D2R–/– mice. Each image is representative of staining patterns seen in specimens from at least three independently examined mice. VEGF is visualized in the red immunofluorescent channel and the pituitary hormones or S100 antigen in the green immunofluorescent channel. A, Prolactin. B, GH. C, LH. D, TSH. E, S100. White arrows indicate pituitary cells double stained. Objective lens magnification, x60. F, Areas of peliosis, arrows indicate groups of red blood cells (black and white graph of a confocal image; magnification, x60).
Discussion
VEGF-A plays pivotal roles in the formation of the vascular systems during embryonic development in the regulation of capillary growth in normal and pathological conditions in adults and the maintenance of the normal vasculature (9). Because VEGF is thought to be the most important angiogenic cytokine in cancer and other types of pathological angiogenesis and because it has been related to the antiangiogenic activity of dopamine in endothelial cells (20, 22), we investigated VEGF expression, localization, and function in preadenomatous pituitary tumors of D2R knockout female mice.
Pituitaries from knockout mice were hyperplastic and hypertrophic as previously described (8, 23); they were proliferating as judged by PCNA expression and showed some areas of peliosis. In a previous paper (8), we reported that knockout mice had a markedly increased number of cells containing prolactin. These lactotrophs were hyperstimulated with rapid turnover of prolactin and limited storage capacity.
We found that VEGF expression was increased in pituitaries from D2R knockout female mice when compared with age-matched wild-type female mice. VEGF production has been demonstrated to be stimulated by estrogen in rat pituitaries (14, 29) and the somatolactotroph cell line GH3 (29) as well as human prolactinomas (28). Nevertheless, estrogen levels are not increased in D2R knockout female mice, indicating that increased pituitary VEGF expression is mainly dependent on the lack of dopaminergic control. In experiments with wild-type female mice, we found that prolonged treatment with the D2R antagonist, haloperidol, enhanced pituitary VEGF protein content and prolactin release. This suggests that dopamine acting at the D2R inhibits pituitary VEGF expression. Haloperidol counteracts dopamine inhibition of cAMP formation in the pituitary (30), and it has been described that agents that enhance cAMP levels such as phorbol esters, forskolin, adenosine, or pituitary adenylate cyclase-activating polypeptide-27 induce VEGF mRNA expression in different tissues (17, 18, 19). Furthermore, it has been described that the antiangiogenic activity of dopamine in endothelial cells is related to its inhibition of VEGF-induced phosphorylation of the VEGF receptor (20, 22). Therefore, it is tempting to speculate that normal D2R-mediated inhibition of adenylate cyclase limits VEGF gene expression. On the other hand, treatment of wild-type females with the long-acting D2R agonist cabergoline did not decrease pituitary VEGF expression, even though it lowered prolactin secretion. Because in wild-type female mice the D2R receptor receives a constant dopaminergic input from the hypothalamus, additional D2R stimulation may not modify VEGF levels.
In the normal human pituitary, VEGF has been localized mainly in ACTH, GH, and follicle stellate cells, with lower levels detected in other cell types (31) (32). In bovine and ovine pituitary cells, VEGF was found mainly in follicle stellate cells (33, 34). And in rats VEGF has also been described in a part of the total TSH cells (35) as well as in the lactosomatotroph GH3 pituitary tumor cell line and a follicle stellate cell line (36, 37). Interestingly, we found that the main source of VEGF-A in the hyperplastic pituitary were follicle stellate cells and not lactotrophs. Because D2R receptors have been described in lactotrophs, it may be inferred that a paracrine-derived factor is acting on follicle stellate cells. To this regard it has been described that agents that increase cAMP levels increase VEGF in a follicle stellate cell line (37), and in D2R–/– lactotrophs, dopamine-mediated inhibition of adenylate cyclase is chronically lacking.
In the present and previous papers (3, 38), we described the occurrence of peliosis (extravasated erythrocytes not contained in capillaries) in the pituitaries of the D2R knockout mice. It is interesting to note that an association of peliosis with tumors that secrete VEGF has been set forth and may be linked to its permeabilizing function (reviewed in Ref. 39). Increased peliosis has been related to high VEGF expression in hepatocarcinogenesis (40), spleen damage (39), and a lethal hepatic syndrome in mice (41), associated with angiogenesis.
We next sought to determine whether VEGF had any action on pituitary cell proliferation or prolactin release. VEGF mediates its mitogenic and vasopermeabilizing effects through two tyrosine kinase receptors, VEGF-R1 (or Flt 1) and VEGF-R2 (or KDR, or Flk-1). Expression of these two VEGF receptors exclusively on endothelial cells (42) indicates that this factor should have no direct influence on endocrine cells. VEGF might act on the intrapituitary endothelium, maintaining vascular integrity and stimulating vascular permeability and endothelial cell proliferation. Nevertheless, there is one report of VEGF-R2 expression in pituitary endocrine cells (43). Flk-1 expression was detected in all types of hormone-producing adenohypophyseal cells as well as in GH3 cells but not in folliculostellate cells. We first found that basal proliferation in vitro estimated using three different assays was lower in cells from knockout donors in contrast to their higher PCNA content in vivo. This has also been previously observed using 5-bromo-2'-deoxyuridine immunoreactivity (38) and may probably indicate that the higher basal proliferation index of wild-type lactotrophs in primary culture is likely due to the acute loss of dopamine inhibition. Consistent with this interpretation, we found that differences in proliferation rate between genotypes were evident only in the first hours after plating, and thereafter cells from both groups had a parallel proliferation curve. Furthermore, increased proliferation in vivo may be dependent on a cohort of growth factors available by the angiogenic process that is increased in knockout and not in wild-type mice.
We found that VEGF did not induce pituitary cellular proliferation; moreover, a prolactin-releasing effect could be evidenced only if cells were pretreated with estrogen. The first result is consistent with several reports that claim that VEGF is a potent mitogen for vascular endothelial cells derived from arteries, veins, and lymphatics but that it is devoid of consistent and appreciable mitogenic activity for other cell types (42). In fact, the denomination of VEGF was proposed to emphasize such narrow target cell specificity.
The prolactin-releasing effect of VEGF has not been described to date. This effect was evidenced only under an estrogenic environment. To this respect, it has been conclusively described that estradiol modifies lactotroph sensitivity to physiological stimulators and inhibitors of prolactin secretion (44). Therefore, increased pituitary VEGF expression may not be important for cellular proliferation of endocrine cells per se, even though it may enhance the prolactin secretory capacity of the gland. On the other hand, increased VEGF may act in adjacent endothelial cells and participate in the angiogenic process that increases the availability of different growth factors and mitogens.
To support this idea, we found that CM from the hyperplastic pituitaries (D2R–/–) was able to induce proliferative changes in HUVECs (this process being mandatory for angiogenesis). The proliferating effect was in part evoked by secreted VEGF, as shown by immunoneutralization experiments. This probably indicates that pituitary-secreted VEGF accumulates in the target endothelial cells in which it may act in a paracrine manner enhancing vessel proliferation.
We also wanted to determine whether estradiol increased VEGF expression in our model but found no significant effect. This lack of effect of estrogen was not related to an alteration of its pituitary receptor, which was increased in female knockouts and was down-regulated by estrogen pretreatment in both genotypes. Estradiol did not increase serum prolactin levels either and even decreased serum and in vitro prolactin levels. This result is consistent with findings by Sinha and Gilligan (45) and our own previous results with strain C57BL/6 mice (4). One possibility is that estradiol may interfere with the actions of hypothalamic-releasing factors leading to storage of prolactin within the lactotropes. Our present results of prolactin inhibition by estradiol in vitro suggest that the steroid interferes directly with lactotrope function. It is feasible that estradiol may alter proteolytic cleavage or other posttranslational modifications of prolactin that could inhibit its secretion. Furthermore, estrogen inhibition of prolactin secretion is strain specific because we showed that estradiol administered under the same conditions in 129S6 mice did cause an increase in serum prolactin levels (4). In addition, other investigators have reported that C57Bl/6J mice are relatively refractory to estradiol-induced pituitary tumorigenesis (46).
In conclusion, we describe that pituitary VEGF expression is increased in female mice lacking dopamine D2Rs. Even though VEGF does not promote pituitary cellular proliferation in vitro, as it does in endothelial cells, it may be critical for effective tumor angiogenesis, which is fundamental for pituitary hyperplasia, and furthermore, it may participate in increased prolactin secretion. Numerous growth factors and their receptors have been identified in the anterior pituitary. It has been postulated that many of these locally produced growth factors may modulate growth function of the pituitary by auto/paracrine mechanisms (47). To our knowledge, this is the first report of dopaminergic control of VEGF expression in the pituitary and may be important in the clinical action of dopaminergic agents. Furthermore, we believe that VEGF and its receptor may become important therapeutic tools in dopamine-resistant prolactinomas.
Acknowledgments
We thank NIDDK’s National Hormone and Pituitary Program and Dr. A. F. Parlow for the prolactin RIA kit.
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Address all correspondence and requests for reprints to: D. Becú-Villalobos, Instituto de Biología y Medicina Experimental, Consejo Nacional de Investigaciones Científicas y Técnicas, V. Obligado 2490, 1428 Buenos Aires, Argentina. E-mail: dbecu@dna.uba.ar.
Abstract
Vascular endothelial growth factor (VEGF)-A is an important angiogenic cytokine in cancer and pathological angiogenesis and has been related to the antiangiogenic activity of dopamine in endothelial cells. We investigated VEGF expression, localization, and function in pituitary hyperplasia of dopamine D2 receptor (D2R)-knockout female mice. Pituitaries from knockout mice showed increased protein and mRNA VEGF-A expression when compared with wild-type mice. In wild-type mice, prolonged treatment with the D2R antagonist, haloperidol, enhanced pituitary VEGF expression and prolactin release, suggesting that dopamine inhibits pituitary VEGF expression. VEGF expression was also increased in pituitary cells from knockout mice, even though these cells proliferated less in vitro when compared with wild-type cells, as determined by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium proliferation assay, proliferating cell nuclear antigen expression, and [3H]thymidine incorporation. In contrast to other animal models, estrogen did not increase pituitary VEGF protein and mRNA expression and lowered serum prolactin secretion in vivo and in vitro in both genotypes. VEGF (10 and 30 ng/ml) did not modify pituitary cell proliferation in either genotype and increased prolactin secretion in vitro in estrogen-pretreated cells of both genotypes. But conditioned media from D2R–/– cells enhanced human umbilical vein cell proliferation, and this effect could be partially inhibited by an anti-VEGF antiserum. Finally, using dual-labeling immunofluorescence and confocal laser microscopy, we found that in the hyperplastic pituitaries, VEGF-A was mostly present in follicle-stellate cells. In conclusion, pituitary VEGF expression is under dopaminergic control, and even though VEGF does not promote pituitary cellular proliferation in vitro, it may be critical for pituitary angiogenesis through paracrine actions in the D2R knockout female mice.
Introduction
THE DOPAMINE D2 receptor (D2R) is the predominant dopamine receptor subtype in the anterior pituitary and mediates dopamine’s inhibitory actions on lactotrophs (1, 2). Recently the importance of D2R in maintaining normal lactotroph function has been clearly demonstrated. Mice lacking D2R generated by homologous recombination in embryonic stem cells display chronic hyperprolactinemia and lactotroph hyperplasia (3, 4). Pituitary adenoma growth, as with all tumors, depends on adequate vascularization. In rats it has been shown that estrogen-induced prolactin-secreting pituitary tumors are highly angiogenic (5), and furthermore tumor growth can be blocked by antiangiogenic agents (6, 7). In D2R knockout mice, prominent vascular channels have been described in the hyperplastic pituitaries as well as extravasated red blood cells or peliosis (8).
Cytokines and growth factors are important modulators of angiogenesis. One of these factors is the vascular endothelial growth factor-A, (VEGF) a dimeric N-glycoprotein of relative molecular mass of 43–46 kDa, formerly described as a permeability factor. VEGF is a potent mitogen for micro- and macrovascular endothelial cells derived from arteries, veins, and lymphatics but not for other cell types (9). Enhanced VEGF expression has been associated with several human vascular tumors, including brain, colon, gastrointestinal tract, ovary, breast, and others (9). Furthermore, this glycoprotein is also abundantly expressed and/or secreted by most animal tumors. Moreover, studies on tumor angiogenesis in nude mice indicate that VEGF expression is critical for effective tumorigenesis and tumor angiogenesis (10, 11).
VEGF has been detected in all types of human pituitary adenomas, primarily in those of somatotrophic or corticotrophic type (12, 13). Furthermore, increased concentrations of VEGF and the VEGF receptor (VEGF-R)2 (KDR, or Flk-1) have previously been reported in rat pituitary tumors (14, 15). The VEGF system plays a crucial role not only in the regulation of tumor angiogenesis during the development of estrogen-induced prolactin secreting pituitary tumors (14) but also in the formation of pituitary portal vessels during fetal life and in the maintenance of their differentiated state in adult animals (16).
The participation of VEGF in pituitary hyperplasia in D2R knockout mice has not been described to date. Even though these animals have low serum estrogen, the loss of the dopamine inhibitory control on lactotrophs may result in a permissive environment for stimulatory factors to function unopposed, leading to lactotroph proliferation, angiogenesis, and tumor development.
Consistent with the presence of activator protein-1 and -2 sites in the VEGF gene promoter, phorbol esters and forskolin that activate adenylate cyclase induce VEGF mRNA expression (17). Besides, several reports have shown that increases in cAMP production stimulate VEGF gene expression (18) (19). In the pituitary of the D2R knockout mice, the lack of action of dopamine on its receptor prevents physiological adenylate cyclase inhibition. Therefore, it was of interest to study the expression levels of VEGF in pituitary cells from D2R knockout mice. Furthermore, in endothelial cells it has been described that dopamine has antiangiogenic activity mediated through the D2R, inhibiting malignant tumors as well as the vascular permeabilizing and angiogenic activities of VEGF (20). Another link of the dopaminergic function and VEGF expression has been reported in two outbred lines of Wistar rats, which present high and low dopaminergic reactivity, respectively. VEGF expression was reduced in the first group, which was more resistant to tumor implantation, and developed significantly fewer lung metastases (21). Finally, in gastric cancer tissues, a low nontoxic dose of dopamine significantly retarded tumor angiogenesis by inhibiting VEGF-R2 phosphorylation within the tumor endothelial cells expressing D2Rs (22).
Therefore, in view of the angiogenic properties of VEGF as well as its relation to dopamine activity in other tissues, we decided to analyze its expression, localization, and regulation by estrogen in pituitary hyperplasia in the D2R knockout female mouse. In addition, we studied the effects of VEGF on prolactin secretion and pituitary cell proliferation in wild-type and D2R knockout female mice.
Materials and Methods
Animals
D2 dopamine receptor knockout mice (official strain designation B6; 129S2-Drd2tm1low by the Induced Mutant Resource at The Jackson Laboratory, Bar Harbor, ME), generated by targeted mutagenesis of the D2R gene in embryonic stem cells (3, 8) were used. The original F2 hybrid strain (129S2/Sv X C57BL/6J) containing the mutated D2R allele was backcrossed for eight generations to wild-type C57BL/6J mice. Mutant and wild-type mice were generally the product of heterozygote crossings, and in all cases sibling controls were used. Female mice were housed in groups of four or five with mixed genotypes in an air-conditioned room with lights on at 0700 h and off at 1900 h. They had free access to laboratory chow and tap water. Wild-type, heterozygous and knockout mice were identified by PCR of genomic DNA, as previously described (23). Animals were used at 8–10 months, and pituitaries from knockout females were hyperplastic at this moment. All experimental procedures were reviewed and approved by the Institutional Animal Care and Use Committee of the Instituto de Biología y Medicina Experimental, Buenos Aires (Division of Animal Welfare, Office for Protection of Research Risks, National Institutes of Health, A#5072–01).
Drugs
Unless specified, all chemicals were purchased from Sigma (St. Louis, MO).
In vivo experiments
Wild-type 8-month-old female mice were divided in groups and treated with castor oil (controls) or haloperidol-decanoate (HALOPIDOL; Janssen-Cilag, Beerse, Belgium), a long-acting D2 antagonist, in a dose of 5 mg/kg, sc, for 3 wk, one injection per week, or 1.2 mg/kg ip for 7 d, one injection per day.
Two other groups of wild-type female mice were injected either with saline solution (controls) or cabergoline (0.5 mg/kg sc; Beta Laboratories, Buenos Aires, Argentina) for 3 wk, two injections per week. Other groups were treated with estradiol-valerate (Progynon Depot; Schering, Buenos Aires, Argentina), 0.2 mg/kg sc or castor oil for controls, 72 and 24 h before sampling. After treatment blood was collected by decapitation. Sera were kept at –20 C until RIAs were performed. Pituitaries were excised as described below for Western blot analysis.
Cell dispersion and culture
Anterior pituitaries from 8- to 10-month-old female wild-type and knockout mice were weighed and dissociated into single cells. Anterior pituitaries were placed in chambers containing freshly prepared Krebs-Ringer bicarbonate buffer without Ca2+ or Mg2+. Buffer contained 14 mM glucose, 1% BSA, 2% MEM amino acids, 1% MEM vitamins (Life Technologies, Inc., Buenos Aires, Argentina), and 2 mM glutamine and was previously gassed during 15 min with 95% O2-5% CO2 and adjusted to pH 7.35–7.40. Buffer was filtered through a 0.45-μm pore diameter membrane (Nalgene, Rochester, NY). Pituitaries were washed three times with Krebs-Ringer bicarbonate buffer and then cut into 1-mm pieces. Fragments obtained were washed and incubated in the same buffer containing 0.5% trypsin for 30 min at 37 C in 95% O2-5% CO2, followed by 2 additional min with 50 μl deoxyribonuclease I (1 mg/ml; Worthington Biochemical Corp., Lakewood, NJ). Digestion was ended by adding 1 mg/ml lima bean trypsin inhibitor. Fragments were disassociated to single cells by gentle trituration through Pasteur pipettes. The resulting suspension was filtered through a nylon gauze (160 μm pore size) and centrifuged 10 min at 1000 x g. Before centrifugation, an aliquot of cellular suspension was taken to quantify pituitary cell yield, using a Neubauer chamber. Viability of cells, determined by Trypan Blue exclusion, was always greater than 90%. Cells (35,000, 250,000, or 375,000 cells/well, depending on the type of assay) were cultured for 5 d in DMEM, 10% horse serum, 2.5% fetal bovine serum (FBS) with our without 10–8 M 17?-estradiol. Cells were then washed and stimulated with 10 or 30 ng/ml recombinant human VEGF-A for 48 h in DMEM 0.5% BSA medium, without serum (with our without estradiol). Cell culture was performed as previously described (24).
Conditioned media (CM) were obtained after culturing 250,000 or 375,000 cells in 24-well plaques for 5 d in the presence of 2.5% FBS (Life Technologies), followed by 2 d without serum, as above described. CM (800 μl) were collected and cells were counted. For RIA assays CM was concentrated (1:10) using a lyophilizer.
RIAs
Prolactin was measured by RIA using a kit provided by the National Institute of Diabetes and Digestive and Kidney Diseases [NIDDK; Dr. A. F. Parlow, National Hormone and Pituitary Program (NHPP), Torrance, CA]. Assays were performed using 10 μl serum in duplicate or the adequate quantity of diluted medium from cultured cells. Results are expressed in terms of mouse prolactin RP3. Intra- and interassay coefficients of variation were 7.2 and 12.8%, respectively.
VEGF
A RIA was developed according to the method described by Anthony et al. (25). Recombinant human VEGF165, the main form of VEGF-A, was used as standard and tracer. VEGF was labeled with 125I (NEN Life Science Products, Inc., Boston, MA) using a modified chloramine T method for iodination. Briefly, 2 μg VEGF was iodinated with 0.7 mCi 125I in 0.5 M phosphate buffer in the presence of 16 μg chloramine T for 45 sec. Reaction was stopped with sodium metabisulfite and the reaction transferred to a Biogel P10 column (Bio-Rad Laboratories, Hercules, CA) previously blocked with 2% BSA in 0.01 M phosphate buffer and 50 mM EDTA. RIA incubation mixtures consisted of 200-μl aliquots of standard (36 to 0.28 ng/tube) or samples in assay buffer [200 mg/liter protamine sulfate, 4.14 g/liter sodium phosphate (monobasic), 0.05% Tween 20 (Promega, Madison, WI), 0.02% sodium azide, and 0.01 M EDTA, 200 μg/ml heparin (Gibco, Buenos Aires, Argentina) (pH 7.5)] containing 100 μl of radioactive tracer 10,000 cpm and 100 μl of rabbit polyclonal VEGF antiserum (1:5,000, sc-507; Santa Cruz Biotechnology, Santa Cruz, CA). After overnight incubation at 4 C, 0.5 ml of 0.01 M PBS containing 5% polyethylene glycol 6000 (Merck-Schuchardt, Hohenbrunn, Germany), 1% (vol/vol) sheep antirabbit -globulin, and 0.05% normal rabbit serum were added to the tubes. After a 2-h incubation at room temperature and an hour at 4 C, tubes were centrifuged at 2000 x g for 30 min at 4 C. The supernatant was aspirated and the radioactivity in the remaining pellet was measured. Assay sensitivity was 0.6 ng/tube. The intraassay coefficient of variation was 10%, and parallelism was obtained between diluted conditioned media from mouse pituitaries and standard human curve. No cross-reactivity was found with basic fibroblast growth factor (FGF) or IGF-I.
Western blot
Anterior pituitaries were homogenized in 80 μl ice-cold buffer containing 60 mM Tris-HCl, 1 mM EDTA (pH 6.8), and a mix of proteases inhibitors in a handheld microtissue homogenizer. The homogenate was then centrifuged at 800 x g for 5 min at 4 C. An aliquot of supernatant was taken to quantify proteins by the Lowry method. Thirty micrograms of proteins in 10 μl of buffer and 60 mM Tris HCl (pH 6.8) were mixed with 10 μl 2 x sample buffer [60 mM Tris-HCl, 4% sodium dodecyl sulfate, 20% glycerol, 0.02% bromophenol blue, and 50 mM dithiothreitol (pH 6.8)]. Samples were sonicated during 20 sec and heated 5 min at 95 C and subjected to 12% SDS-PAGE. The gel was then blotted onto a nitrocellulose membrane (Bio-Rad) and probed with the corresponding primary antibody followed by a secondary antibody conjugated with horseradish peroxidase. Polyclonal rabbit VEGF antibody (1:1000, sc-507; Santa Cruz Biotechnology) was used. This antibody recognizes the 189-, 165-, and 121-amino acid splice variants of VEGF-A. The visible band corresponded to VEGF165, and in some cases a doublet of this band was observed. Proliferating cell nuclear antigen [PCNA (FL-261): sc-7907] and estrogen receptor- [ER (MC-20), sc-542] antibodies were purchased from Santa Cruz Biotechnologies. Monoclonal mouse actin antibody (Ab-1) was purchased from Lab Vision Co. (Fremont, CA). Immunoreactive proteins were detected by enhanced chemiluminescence (Amersham, Buenos Aires, Argentina). For repeated immunoblotting, membranes were incubated in stripping buffer [62.5 mM Tris, 2% sodium dodecyl sulfate, and 100 mM mercaptoethanol (pH 6.7)] for 40 min at 50 C and reprobed. Band intensities were quantified using the ImageQuant software.
Preparation of pituitary RNA
Total RNA was isolated from anterior pituitaries using TRIzol reagent (Gibco). Each gland was homogenized in 100 μl TRIzol, sonicated for 10 sec, and incubated at room temperature for 5 min. Chloroform (20 μl) was added, samples were shaken vigorously, and after 5 min of incubation at room temperature, they were centrifuged at 12,000 x g for 15 min at 4 C. Isopropanol (50 μl) was added to the supernatant to precipitate the RNA. After a 10-min incubation at room temperature, samples were centrifuged at 12,000 x g for 10 min at 4 C, supernatants discarded, and their pellets washed with 100 μl of 70% ethanol. The resulting precipitates were resuspended in 5 μl diethylpyrocarbonate-treated water. RNA was quantified by UV spectrophotometry and its integrity checked by gel electrophoresis.
Semiquantitative RT-PCR
Total RNA (1.5 μg) was reverse transcribed in a reaction mixture containing 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol, 1 mM deoxynucleotide triphosphates, 8 U Rnase inhibitor (Promega), 1 μg of random hexamers (Biodynamics SRL, Buenos Aires, Argentina), and 200 U Moloney murine leukemia virus transcriptase (Invitrogen Life Technologies, Buenos Aires, Argentina) in a final volume of 20 μl. After incubation at 37 C for 60 min, samples were heated for 10 min at 70 C to inactivate the transcriptase. The product was amplified with VEGF and glycerol-3-phosphate dehydrogenase (G3PDH) sense and antisense primers in a reaction mixture (30 μl) containing 20 mM Tris-HCl (pH 8.4), 50 mM KCl, MgCl2 (see Table 1), 0.2 mM deoxynucleotide triphosphates, 0.5 μM of each primer, and Taq DNA polymerase (Invitrogen Life Technologies), using an Eppendorf thermal cycler.
TABLE 1. Description of primers used and PCR conditions
In Table 1, primer sequences and conditions of PCR amplification are detailed. For VEGF, primers were chosen to detect all splice variants of VEGF-A because they were directed against the common area of the cDNA (26). Common steps were a hot start step of 3 min at 95 C, followed by n cycles of denaturation at 94 C for 60 sec, annealing for 60 sec, and extension at 72 C for 50 sec, with a final elongation step of 5 min at 72 C.
Preliminary experiments using various RNA concentrations and cycle numbers confirmed that these PCRs were performed within the linear phase of the PCR amplification reaction. Ten microliters of amplified mixture were mixed with 1 μl of sample buffer (25% bromophenol blue, 30% glycerol) and analyzed by 1.8% agarose gel electrophoresis. The amplified DNA bands were detected by ethidium bromide staining. Densitometric analysis was conducted using the Scion Image software and intensity values of VEGF PCR products were normalized to the corresponding G3PDH products.
DNA synthesis in pituitary cells in culture
Culture procedure was the same as described above. [3H]thymidine (0.2 μCi/well, 87.7 Ci/mmol, NEN Life Science Products) was added to cultures. After 24 h. of incubation, medium was discarded and the cells were removed and lysed by treatment with 0.05% trypsin and 0.02% EDTA in deionized water. The reaction was stopped 20 min later by filtering under vacuum through GF/C filters (Whatman, Middlesex, UK) using the Cell Harvester 8 (Nunc, Glastrup, Denmark). After five washes with deionized water, the filters were placed in plastic vials with 3 ml scintillation solution and radioactivity counted in a Beckman counter. Each experiment was repeated six times.
Cell proliferation assay
Proliferation of anterior pituitary cells was also colorimetrically determined at 490 nm using a commercial proliferation assay kit (CellTiter 96 AQueous nonradioactive cell proliferation assay; Promega). After incubation with various concentrations of VEGF-A for 48 h, cells in a 96-well plate were incubated with 333 mg/liter 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) and 25 μM phenazine methosulfate solution for 0.5, 1, and 2 h at 37 C in a humidified, 50 ml/liter CO2 atmosphere. The absorbance of soluble formazan produced by cellular reduction of MTS was measured at 490 nm using an ELISA reader (Sensident scan; Merck). In each experiment four to six mice of each genotype were used, experiments were repeated four times, and each had quadruplicate samples.
To evaluate the time curve of pituitary cell proliferation comparatively between genotypes, cells were dispersed on d 1 and plated in quadruplicate in eight individual 96-well plates. Proliferation was assayed at time 0, 5 h, and 1, 2, 3, 4, 5, and 7 d as described above. In one set of experiments on d 5, medium was refreshed, and cells were cultured for 4 additional days in medium with serum. Experiment was repeated three times.
Human umbilical cord vein endothelial cell (HUVEC) culture and proliferation assay
Endothelial cells were isolated from HUVECs by enzymatic digestion with collagenase as previously described (27). HUVECs were cultured in T75 flasks in M199 supplemented with 20% FBS, growth factors, and 50 μg/ml gentamicin and were maintained at 37 C in a fully humidified atmosphere of 5% CO2 in the air. The culture medium was changed every 72 h, and HUVEC confluent cultures were washed twice with PBS, released with 0.05% (wt/vol) trypsin and 5 mM EDTA and subcultured. Cell proliferation studies were carried out using endothelial cells at passages 6–9.
The proliferation of HUVECs was measured by [3H]thymidine incorporation. HUVECs were harvested with trypsin/EDTA and suspended in M199 (supplemented with 20% FBS and 50 μg/ml gentamicin) at a density of 25,000 cells/ml and then seeded into a 96-well plate (100 μl per well: 2500 cells/well) and incubated for 2 h for attachment. Then 50 μl of the same medium with murine VEGF-A (final concentration 10 ng/ml), basic FGF (final concentration 2 ng/ml), epidermal growth factor (EGF, 1 ng/ml), or various CM were added alone or with 2.5 μg/ml polyclonal antibody against VEGF-A. They were incubated for 24 h before adding 5 μCi/ml [3H]thymidine. After 48 h incubation, the assay was ended by adding 50 μl guanidine-HCl, and the cells were lysed by a freezing-defrost cycle. The DNA was harvested in Whatman GF/C filters by using an 8-well harvester (Cell Harvester 8; Nunc), and 1 ml scintillation solution (OptiPhase Hifase 3) was added. The [3H]thymidine incorporation was measured by using a liquid scintillation counter.
Double-labeling immunofluorescence and confocal laser microscopy
Double-labeling immunofluorescence was applied to specifically identify the cell type(s) expressing VEGF-A. Double immunostaining was performed on paraffin-embedded sections of D2R knockout mice of the 129S6 and C57BL/6 congenic strains. We combined chicken anti-VEGF antibody with rabbit polyclonal antibodies against pituitary hormones or S-100 protein, a marker of folliculostellate cells. Specifics of the various antisera employed were as follows: chicken polyclonal to VEGF (dilution 1:100, ab 14078; Abcam, Cambridge MA), rabbit antisera directed against mouse prolactin (dilution 1:500; NHPP, NIDDK-AFP-107120402), mouse GH (dilution 1:1000, NHPP, NIDDK-AFP-5672099), rat TSH (?-TSH) (dilution 1:500; NHPP, NIDDK-AFP-1274789), rat LH (?-LH) (dilution 1:1,200; NHPP, NIDDK-AFP-571292393), and bovine S-100 protein (1:200, Ab2; Lab Vision). After rinsing in PBS, the double-stained sections were incubated at room temperature for 2 h with fluorescein isothiocyanate goat antirabbit IgG (dilution 1:100; Zymed Laboratories Inc., San Francisco, CA) and Texas red-X-conjugated goat antichicken IgY (dilution 1:100, Sc-2994; Santa Cruz Biotechnologies). After rinsing in PBS, the sections were mounted in Vectashield (Vector Laboratories, Burlingame, CA) to prevent fading of the immunofluorescence reaction. Sections were examined on a C1 Plan Apo x60/1.4 oil confocal laser-scanning system (Nikon, Tokyo, Japan). The excitation wavelength was 488 nm for fluorescein isothiocyanate and 543 nm for Texas red-X-induced fluorescence. Specificity studies were carried out by omitting primary antisera or preabsorbing the primary antisera with homologous antigen excess; all showed the absence of the fluorescent signal.
Statistical analyses
Results are expressed as means ± SE. VEGF expression, ER expression, and prolactin release in vivo and in vitro were analyzed by two-way ANOVA for independent measures for the effects of genotype and estrogen treatment. PCNA expression in vivo, VEGF-A release in vitro, PCNA expression in vitro, 3(H)-thymidine uptake, MTS assay, and haloperidol and cabergoline effects were analyzed by one-way ANOVA. Effect of CM on proliferation of HUVECs, and proliferation time curve were analyzed by two-way ANOVA for repeated measures. In all cases, if F of interaction was found significant, individual means were compared by Tukey’s honest significant difference or Fisher’s protected least significant difference tests; if it was not significant, groups of means were analyzed by the same tests. P < 0.05 was considered significant.
Results
When compared with female wild-type mice, pituitaries from female D2R knockout mice had increased VEGF concentration (normalized to actin content, Fig. 1A; P = 0.0036) as well as greater VEGF mRNA expression (normalized to the housekeeping gene G3PDH, Fig. 1B; P = 0.043). Enhanced PCNA concentration was also found in pituitaries from female knockout mice (Fig. 1C; P = 0.026) in concordance with increased pituitary weight as previously described (23) (pituitary weight in milligrams ± SE was 7.03 ± 1.39 and 2.51 ± 0.34 for knockout and wild-type mice, respectively; P = 0.011).
FIG. 1. A, Comparative pituitary VEGF-A content (evaluated by Western blot) in wild-type (+/+, open bars) and D2R knockout (–/–, filled bars) female mice. For each sample, arbitrary units of band intensities for VEGF-A were divided by band intensities for the respective actin and compared with those of wild-type females (considered 100%) in each series of experiments. *, P < 0.05 vs. +/+; n = 18 and 15, respectively. For this and following figures, results shown are means ± SE. B, Densitometric analysis of pituitary VEGF-A RT-PCR products in wild-type (open bars) and D2R knockout (filled bars) female mice. For each sample, intensity units of the VEGF band were normalized to those of the respective G3PDH band. *, P < 0.05 vs. +/+; n = 5 and 6. C, Pituitary PCNA content (evaluated by Western blot) in wild-type (+/+, open bars) and D2R knockout (–/–; filled bars) female mice. For each sample, arbitrary units of band intensities for PCNA were divided by band intensities for the respective actin and compared with those of wild-type females (considered 100%) in each series of experiments. *, P < 0.05 vs. +/+; n = 9 and 11, respectively. Below each graph representative bands are depicted.
A group of female wild-type mice was treated for 1 or 3 wk with a long-acting D2R antagonist, haloperidol-decanoate. We found that the longer treatment evoked a significant increase in pituitary VEGF concentration (Fig. 2A; P = 0.0029). As expected, prolactin levels also rose in this group (Fig. 2C; P = 0.0064). On the other hand, a 3-wk treatment with the D2R agonist cabergoline lowered prolactin levels in female wild-type mice (Fig. 2D; P = 0.0012) but did not significantly decrease pituitary VEGF expression (Fig. 2B; P = 0.57).
FIG. 2. Left panels, Effect of in vivo haloperidol treatment in wild-type females on pituitary VEGF-A content, normalized to the respective actin content of the sample (A) and serum prolactin levels (nanograms per milliliter) (C). Oil-injected females (oil) were considered as 100% in A. hal1, One week of haloperidol decanoate (see Material and Methods); hal3, 3 wk of haloperidol deca-noate treatment (n = 10, 7, and 6, respectively). Right panels, Effect of prolonged in vivo cabergoline (CB) treatment in wild-type females on pituitary VEGF-A content (B) and serum prolactin levels (nanograms per milliliter) (D) (n = 6 for each group). *, P < 0.05 vs. control (SAL). Below A and C are representative bands.
We next determined VEGF cellular expression and secretion, prolactin secretion, and basal cell proliferation in cultured pituitary cells from wild-type and knockout female mice. VEGF concentration in cells, measured by Western blot and normalized to actin content, was also increased in pituitary cells from knockout females (Fig. 3A; P = 0.018). Furthermore, in conditioned media from cells from knockout mice, higher VEGF and prolactin, measured by RIA, were found (Fig. 3, B and C; P = 0.043 and 0.0099, respectively).
FIG. 3. A, VEGF-A content (Western blot) in pituitary cells cultured in vitro (n = 4). B,VEGF-A release (nanograms per milliliter, RIA) from pituitary cells cultured in vitro (n = 6). C, Prolactin release in vitro (n = 4). *, P < 0.05 vs. respective wild type.
However, the rate of cell proliferation in this group was lower when compared with wild-type mice as determined using the MTS proliferation assay, PCNA expression by Western blot, and [3H]thymidine incorporation (Fig. 4, A–C; P = 0.000155, 0.039, and < 0.00001 for the three assays, respectively). Using identical conditions to the experiments described above (cells were cultured for 5 d with serum and serum starved for 2 additional days), we performed a proliferation time curve with cells from both genotypes and found that during the first 5 h, cells from wild-type pituitaries had a higher proliferation rate in comparison with their knockout counterparts; thereafter cells from both genotypes exhibited a parallel proliferation curve, with higher number of cells (as assayed by the MTS test) in wild-types (Fig. 4D). In a new set of experiments, at 5 d medium was changed and cells were cultured for 4 additional days in the presence of serum; differences in MTS assay were maintained (OD ± SE for wild-type and knockout: 0.0169 ± 0.001 and 0.068 ± 0.011, respectively; P < 0.01).
FIG. 4. A, MTS proliferation assay. Results are expressed as OD at 490 nm (n = 4). B, PCNA content in cultured pituitary cells (n = 5). C, [3H]thymidine incorporation in cultured pituitary cells (n = 9). D, Proliferation time curve (MTS assay; 35,000 original cells per well; n = 4). *, P < 0.05 vs. respective wild type.
As described, whole CM was first analyzed for the presence of VEGF-A by RIA. The results showed that VEGF was present in CM from both genotypes and that the concentration was higher in CM from D2R–/– cells (Fig. 3B). We next tested the effect of CM on DNA synthesis in endothelial cells by the measurement of [3H]thymidine incorporation into DNA in confluent quiescent HUVECs in the presence and absence of whole CM from anterior pituitary cells from both genotypes. The addition of CM from both genotypes to quiescent cells led to a significant increase in DNA synthesis in HUVECs, but the effect was significantly higher in CM from D2R–/– cells (P = 0.0024 vs. wild-type cells, Fig. 5A). There was a significant interaction for the effects of anti-VEGF pretreatment and group F(2, 9) = 4.35, P = 0.048. The proliferating effect was decreased in the presence of anti-VEGF in CM from D2R–/– (P = 0.038) and not from wild-type cells (P = 0.23). As an internal control of proliferation, we determined that addition of VEGF (10 ng/ml), EGF (1 ng/ml), and basic FGF (2 ng/ml) markedly increased [3H]thymidine incorporation, and only the effect of VEGF was blocked by anti-VEGF pretreatment (Fig. 5B).
FIG. 5. Effect of CM obtained from pituitary cells from knockout (KO) or wild-type (WT) female mice on HUVEC proliferation. A, HUVECs were grown to confluence and then exposed to CM or serum-free DMEM in presence or absence (buffer) of anti-VEGF (2.5 μg/ml). Proliferation was estimated as the incorporation of [3H]thymidine into DNA (counts per minute). , P = < 0.05 vs. WT. *, P < 0.05 vs. respective buffer (n = 4). B, Internal controls of HUVEC proliferation, murine VEGF-A (10 ng/ml), EGF (1 ng/ml), FGF-2 (2 ng/ml), n = 3.
We tested whether estrogen treatment in vivo could increase pituitary VEGF concentration in both genotypes. We found that estrogen did not increase VEGF concentration (protein or mRNA) in D2R knockout mice (Fig. 6, A and B; effect of estrogen on VEGF protein or mRNA was P = 0.78 and 0.27, respectively). This was not due to lack of pituitary ER, which was increased in knockout mice and could be down-regulated by estrogen treatment (Fig. 6C; P = 0.044 and 0.00070 for the effects of genotype and estrogen treatment; interaction P = 0.21).
FIG. 6. Effect of estradiol valerate (estrogen) treatment in vivo (0.2 mg/kg sc or castor oil for controls 72 and 24 h before sampling) on pituitary VEGF-A content evaluated by Western blot and normalized to the respective actin content of the sample (A); pituitary VEGF mRNA evaluated by RT-PCR and normalized to respective G3PDH mRNA (B); pituitary ER by Western blot normalized to actin content (C); and serum prolactin (nanograms per milliliter) (D). E, Effect of 10–8 M estradiol in vitro on prolactin release in cultured pituitary cells from donor wild-type and knockout females. *, P < 0.05 vs. respective oil-treated control. , P < 0.05 vs. treatment-matched wild-type mice. N = 18, 14, 15, and 19 (A); 5, 4, 6, 6 (B); 5, 7, 6, and 7 (C); 29, 25, 23, and 22 (D); 4 for each group (E).
VEGF protein and mRNA concentration were not increased either by estrogen in wild-type C57BL/6 mice, in contrast to other experimental models (14, 28). But also in contrast to other data reported, estrogen lowered serum prolactin release in vivo and prolactin secretion in vitro in both genotypes [Fig. 6, D and E; P interaction (genotype x estrogen) = 0.12 and 0.96; P effect estrogen = 0.0015 and 0.00015 for in vivo and in vitro, respectively].
Because pituitary VEGF was increased in D2R knockout females, we evaluated whether VEGF could affect pituitary cellular proliferation and/or prolactin secretion in both genotypes in the presence or absence of estrogen in vitro. VEGF (10 and 30 ng/ml) did not modify cell proliferation in either genotype, treated or not with estrogen (Fig. 7, A and B; P = 0.74 and 0.43 for the effect of drug in wild-type and D2R knockout, respectively) in contrast to its effect on endothelial cells. Similar results were obtained with PCNA/actin expression in buffer-pretreated cells (data not shown). Even though VEGF did not affect prolactin secretion in buffer-pretreated cells of either genotype, it significantly increased prolactin release in both genotypes when estrogen was added to the medium (Fig. 8, A and B; P = 0.039 and 0.027, VEGF 10 ng/ml vs. buffer for wild-type and knockout, respectively).
FIG. 7. MTS proliferation assay in cultured pituitary cells from wild-type (A) and knockout mice (B). Cells were pretreated with buffer (empty bars) or 10–8 M estradiol (5 d, hatched bars) and then stimulated with buffer (ctrol) or VEGF (10 or 30 ng/ml, VEGF10 and VEGF30) in buffer with or without estradiol for 48 h. Results are expressed as OD at 490 nm (n = 4 for each group).
FIG. 8. Prolactin release in cultured pituitary cells from wild-type (A) and knockout mice (B). Cells were pretreated with buffer (empty bars) or 10–8 M estradiol (5 d, hatched bars) and then stimulated with buffer (Ctrl) or VEGF (10 or 30 ng/ml, VEGF10 and VEGF30) in buffer with or without estradiol for 48 h. Results are expressed as percent release with respect to buffer-treated cells (n = 4 for each group). P < 0.05 vs. respective genotype- and pretreatment-matched control (Ctrl).
Double-labeling immunofluorescence and confocal laser microscopy was applied to specifically identify the cell type(s) expressing VEGF-A in pituitaries from D2R knockout mice. As shown in Fig. 9, A–E, VEGF-A strongly colocalized with the S-100 protein and not with prolactin, TSH, LH, or GH. Besides, we found histologic evidence of peliosis (Fig. 9E).
FIG. 9. Double-label immunofluorescent staining combined with confocal laser microscopy in the pituitary of D2R–/– mice. Each image is representative of staining patterns seen in specimens from at least three independently examined mice. VEGF is visualized in the red immunofluorescent channel and the pituitary hormones or S100 antigen in the green immunofluorescent channel. A, Prolactin. B, GH. C, LH. D, TSH. E, S100. White arrows indicate pituitary cells double stained. Objective lens magnification, x60. F, Areas of peliosis, arrows indicate groups of red blood cells (black and white graph of a confocal image; magnification, x60).
Discussion
VEGF-A plays pivotal roles in the formation of the vascular systems during embryonic development in the regulation of capillary growth in normal and pathological conditions in adults and the maintenance of the normal vasculature (9). Because VEGF is thought to be the most important angiogenic cytokine in cancer and other types of pathological angiogenesis and because it has been related to the antiangiogenic activity of dopamine in endothelial cells (20, 22), we investigated VEGF expression, localization, and function in preadenomatous pituitary tumors of D2R knockout female mice.
Pituitaries from knockout mice were hyperplastic and hypertrophic as previously described (8, 23); they were proliferating as judged by PCNA expression and showed some areas of peliosis. In a previous paper (8), we reported that knockout mice had a markedly increased number of cells containing prolactin. These lactotrophs were hyperstimulated with rapid turnover of prolactin and limited storage capacity.
We found that VEGF expression was increased in pituitaries from D2R knockout female mice when compared with age-matched wild-type female mice. VEGF production has been demonstrated to be stimulated by estrogen in rat pituitaries (14, 29) and the somatolactotroph cell line GH3 (29) as well as human prolactinomas (28). Nevertheless, estrogen levels are not increased in D2R knockout female mice, indicating that increased pituitary VEGF expression is mainly dependent on the lack of dopaminergic control. In experiments with wild-type female mice, we found that prolonged treatment with the D2R antagonist, haloperidol, enhanced pituitary VEGF protein content and prolactin release. This suggests that dopamine acting at the D2R inhibits pituitary VEGF expression. Haloperidol counteracts dopamine inhibition of cAMP formation in the pituitary (30), and it has been described that agents that enhance cAMP levels such as phorbol esters, forskolin, adenosine, or pituitary adenylate cyclase-activating polypeptide-27 induce VEGF mRNA expression in different tissues (17, 18, 19). Furthermore, it has been described that the antiangiogenic activity of dopamine in endothelial cells is related to its inhibition of VEGF-induced phosphorylation of the VEGF receptor (20, 22). Therefore, it is tempting to speculate that normal D2R-mediated inhibition of adenylate cyclase limits VEGF gene expression. On the other hand, treatment of wild-type females with the long-acting D2R agonist cabergoline did not decrease pituitary VEGF expression, even though it lowered prolactin secretion. Because in wild-type female mice the D2R receptor receives a constant dopaminergic input from the hypothalamus, additional D2R stimulation may not modify VEGF levels.
In the normal human pituitary, VEGF has been localized mainly in ACTH, GH, and follicle stellate cells, with lower levels detected in other cell types (31) (32). In bovine and ovine pituitary cells, VEGF was found mainly in follicle stellate cells (33, 34). And in rats VEGF has also been described in a part of the total TSH cells (35) as well as in the lactosomatotroph GH3 pituitary tumor cell line and a follicle stellate cell line (36, 37). Interestingly, we found that the main source of VEGF-A in the hyperplastic pituitary were follicle stellate cells and not lactotrophs. Because D2R receptors have been described in lactotrophs, it may be inferred that a paracrine-derived factor is acting on follicle stellate cells. To this regard it has been described that agents that increase cAMP levels increase VEGF in a follicle stellate cell line (37), and in D2R–/– lactotrophs, dopamine-mediated inhibition of adenylate cyclase is chronically lacking.
In the present and previous papers (3, 38), we described the occurrence of peliosis (extravasated erythrocytes not contained in capillaries) in the pituitaries of the D2R knockout mice. It is interesting to note that an association of peliosis with tumors that secrete VEGF has been set forth and may be linked to its permeabilizing function (reviewed in Ref. 39). Increased peliosis has been related to high VEGF expression in hepatocarcinogenesis (40), spleen damage (39), and a lethal hepatic syndrome in mice (41), associated with angiogenesis.
We next sought to determine whether VEGF had any action on pituitary cell proliferation or prolactin release. VEGF mediates its mitogenic and vasopermeabilizing effects through two tyrosine kinase receptors, VEGF-R1 (or Flt 1) and VEGF-R2 (or KDR, or Flk-1). Expression of these two VEGF receptors exclusively on endothelial cells (42) indicates that this factor should have no direct influence on endocrine cells. VEGF might act on the intrapituitary endothelium, maintaining vascular integrity and stimulating vascular permeability and endothelial cell proliferation. Nevertheless, there is one report of VEGF-R2 expression in pituitary endocrine cells (43). Flk-1 expression was detected in all types of hormone-producing adenohypophyseal cells as well as in GH3 cells but not in folliculostellate cells. We first found that basal proliferation in vitro estimated using three different assays was lower in cells from knockout donors in contrast to their higher PCNA content in vivo. This has also been previously observed using 5-bromo-2'-deoxyuridine immunoreactivity (38) and may probably indicate that the higher basal proliferation index of wild-type lactotrophs in primary culture is likely due to the acute loss of dopamine inhibition. Consistent with this interpretation, we found that differences in proliferation rate between genotypes were evident only in the first hours after plating, and thereafter cells from both groups had a parallel proliferation curve. Furthermore, increased proliferation in vivo may be dependent on a cohort of growth factors available by the angiogenic process that is increased in knockout and not in wild-type mice.
We found that VEGF did not induce pituitary cellular proliferation; moreover, a prolactin-releasing effect could be evidenced only if cells were pretreated with estrogen. The first result is consistent with several reports that claim that VEGF is a potent mitogen for vascular endothelial cells derived from arteries, veins, and lymphatics but that it is devoid of consistent and appreciable mitogenic activity for other cell types (42). In fact, the denomination of VEGF was proposed to emphasize such narrow target cell specificity.
The prolactin-releasing effect of VEGF has not been described to date. This effect was evidenced only under an estrogenic environment. To this respect, it has been conclusively described that estradiol modifies lactotroph sensitivity to physiological stimulators and inhibitors of prolactin secretion (44). Therefore, increased pituitary VEGF expression may not be important for cellular proliferation of endocrine cells per se, even though it may enhance the prolactin secretory capacity of the gland. On the other hand, increased VEGF may act in adjacent endothelial cells and participate in the angiogenic process that increases the availability of different growth factors and mitogens.
To support this idea, we found that CM from the hyperplastic pituitaries (D2R–/–) was able to induce proliferative changes in HUVECs (this process being mandatory for angiogenesis). The proliferating effect was in part evoked by secreted VEGF, as shown by immunoneutralization experiments. This probably indicates that pituitary-secreted VEGF accumulates in the target endothelial cells in which it may act in a paracrine manner enhancing vessel proliferation.
We also wanted to determine whether estradiol increased VEGF expression in our model but found no significant effect. This lack of effect of estrogen was not related to an alteration of its pituitary receptor, which was increased in female knockouts and was down-regulated by estrogen pretreatment in both genotypes. Estradiol did not increase serum prolactin levels either and even decreased serum and in vitro prolactin levels. This result is consistent with findings by Sinha and Gilligan (45) and our own previous results with strain C57BL/6 mice (4). One possibility is that estradiol may interfere with the actions of hypothalamic-releasing factors leading to storage of prolactin within the lactotropes. Our present results of prolactin inhibition by estradiol in vitro suggest that the steroid interferes directly with lactotrope function. It is feasible that estradiol may alter proteolytic cleavage or other posttranslational modifications of prolactin that could inhibit its secretion. Furthermore, estrogen inhibition of prolactin secretion is strain specific because we showed that estradiol administered under the same conditions in 129S6 mice did cause an increase in serum prolactin levels (4). In addition, other investigators have reported that C57Bl/6J mice are relatively refractory to estradiol-induced pituitary tumorigenesis (46).
In conclusion, we describe that pituitary VEGF expression is increased in female mice lacking dopamine D2Rs. Even though VEGF does not promote pituitary cellular proliferation in vitro, as it does in endothelial cells, it may be critical for effective tumor angiogenesis, which is fundamental for pituitary hyperplasia, and furthermore, it may participate in increased prolactin secretion. Numerous growth factors and their receptors have been identified in the anterior pituitary. It has been postulated that many of these locally produced growth factors may modulate growth function of the pituitary by auto/paracrine mechanisms (47). To our knowledge, this is the first report of dopaminergic control of VEGF expression in the pituitary and may be important in the clinical action of dopaminergic agents. Furthermore, we believe that VEGF and its receptor may become important therapeutic tools in dopamine-resistant prolactinomas.
Acknowledgments
We thank NIDDK’s National Hormone and Pituitary Program and Dr. A. F. Parlow for the prolactin RIA kit.
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