Over expression of vascular endothelial growth factor and its receptor ...

Carcinogenesis vol.18 no.6 pp.1155–1161, 1997

Over expression of vascular endothelial growth factor and its receptor during the development of estrogen-induced rat pituitary tumors may mediate estrogen-initiated tumor angiogenesis

Sushanta K.Banerjee1,3, Dipak K.Sarkar2, Allan P.Weston1, Alok De2 and Donald R.Campbell1 1Cancer

Research Unit, Research Division, V.A. Medical Center, Kansas City, MO and Department of Internal Medicine, University of Kansas Medical Center, Kansas City, Kansas and 2Department of Veterinary and Comparative Anatomy, Pharmacology and Physiology, Washington State University, Pullman, Washington, USA 3To

whom correspondence should be addressed: Molecular Gastroenterology and Pancreatic Cancer Research Unit, V.A. Medical Center, Research Division (151), 4801 Linwood Blvd, Kansas City, MO 64128-2226, USA

Estrogens, which have been associated with several types of human and animal cancers, can induce tumor angiogenesis in the pituitary of Fischer 344 rats. The mechanistic details of tumor angiogenesis induction, during estrogen carcinogenesis, are still unknown. To elucidate the role of estrogen in the regulation of tumor angiogenesis in the pituitary of female rats, the density of blood vessels was analysed using factor VIII related antigen (FVIIIRAg) immunohistochemistry and the expression of vascular endothelial growth factor/vascular permeability factor (VEGF/ VPF) was examined by Western blot and immunohistochemical analysis. The expression of VEGF receptor (VEGFR-2/Flk-1/KDR) was also examined by immunohistochemistry. The results demonstrated that 17β-estradiol (E2) induces neovascularization, as well as the growth and enlargement of blood vessels after 7 days of exposure. The high tumor angiogenic potential was associated with an elevated VEGF/VPF protein expression in the E2 exposed pituitary of ovariectomized (OVEX) rats. VEGF/VPF and FVIIIRAg immunohistochemistry and endothelial specific lectin (UEA1) binding studies, indicate that the elevation of VEGF protein expression initially occurred in both blood vessels and non-endothelial cells. After 15 days of E2 exposure, VEGF/VPF protein expression, in the non-endothelial cell population, sharply declined and was restricted to the blood vessels. The function of non-endothelial-derived VEGF is not clear. Furthermore, immunohistochemical studies demonstrated that VEGFR-2 (flk-1/KDR), expression was elevated significantly in the endothelial cells of microblood vessels after 7 days of E2 exposure. These findings suggest that over expression of VEGF and its receptor (VEGFR-2) may play an important role in the initial step of the regulation of estrogen induced tumor angiogenesis in the rat pituitary. Introduction Estrogens have been associated with several types of human cancers (1) and can induce tumors in a variety of animal models (2). Prolactin-secreting pituitary tumors can be induced in certain strains of rats by prolonged estrogen treatment (3). The mechan*Abbreviations: VEGF/VPF, vascular endothelial growth factor/vascular permeability factor; E2, 17β-estradiol; VEA1, Ulex europaeus agglutinin 1; FVIIIRAg, factor VIII related antigen; OVEX, ovariectomized. © Oxford University Press

ism of pituitary tumor induction by estrogen remains largely unknown. Previous studies have shown that the estrogeninduced prolactin-secreting pituitary tumors, in Fischer rats, are highly angiogenic (4) and tumor growth can be blocked by antiangiogenic agents (5,6). These studies also suggest that the growth of this tumor is angiogenic dependent. However, it is not apparent how estrogen modulates tumor angiogenesis in the anterior pituitary of rats. To date, several tumor angiogenic factors have been identified and characterized (7,8). One of these factors is vascular endothelial growth factor/vascular permeability factor (VEGF/VPF*), a dimeric N-glycoprotein of relative molecular mass 43–46 kDa. It is an important angiogenic factor in various human cancers including human brain (9–11), colonic (12), gastrointestinal tract (13), ovaries (14), breast (15) and other cancers (8). This glycoprotein is also abundantly expressed and/or secreted by most animal tumors examined thus far (7). Moreover, recent studies on tumor angiogenesis in nude mice indicate that VEGF/VPF expression is critical for effective tumorigenesis and tumor angiogenesis (16–19). However, this factor does not induce or promote cellular transformation in vitro (20). VEGF/VPF mediates its mitogenic and vasopermeabilic effects through the two tyrosine kinase family receptors which are VEGF-R1, originally known as Flt 1 (21,22), and VEGF-R2 [also known as KDR (23), Flk-1(24,25)]. Both receptors have been shown to be selectively expressed in endothelial cells (26,27). However, VEGF-R2 was found to mediate essential functions in endothelial cells such as chemotaxis, cytoskeletal recognization and mitogenesis upon VEGF stimulation, whereas VEGF-R1 expressing cells lacked such responses (28). Furthermore, over expression of a dominant-negative VEGF-R2 (flk-1) was found to reduce tumor angiogenesis and growth (29). Taken together, these studies indicate that both VEGF/VPF and its receptor, VEGF-R2, play important roles in the development of tumor angiogenesis and growth. The regulation of VEGF/VPF and its receptor’s expression during normal and pathological conditions is complex and the molecular mechanism(s) involved in these regulatory pathways remain elusive (30). Expression of this growth factor can be induced rapidly by estrogen in the rat uterus (31), mouse ovaries and in endometrial carcinoma cells (30) indicating that VEGF/ VPF expression is elevated in organs actively undergoing neovascularization controlled by estroid hormone (30). Although estrogen regulates VEGF/VPF expression under normal physiolgical conditions in certain tissues, at present it is not known whether prolonged exposure of high levels of estrogen can generate a potent stimulus like VEGF for continual angiogenesis in the target organs during the development of tumors. The goal of the present study was to investigate the changes of VEGF/VPF and VEGF-R2 expression and cell type specific localization in pituitary tissues during the development of estrogen-induced tumor angiogenesis in the anterior pituitary of Fischer 344 rats. Materials and methods Chemicals 17β-Estradiol (E2) and endothelial cell specific peroxidase conjugated Lactin Ulex europaeus agglutinin 1 (UEA1) were purchased from Sigma Chemical

1155

S.K.Banerjee et al. Co., (St. Louis, MO). Immunohistochemical kits, citrate buffer and factor VIII related antigen (FVIIIRAg) were purchased from Zymed Laboratories Inc. (San Francisco, CA). Western blot ECL Cheminufluorescence detection kit was purchased from Amersham Life Science (Cleveland, OH). VEGF polyclonal antibodies were obtained from NeoMarkers (Freemont, CA) and Santa Cruz Biotechnology (Santa Cruz, CA). VEGF receptor KDR/Flk-1 (VEGFR-2) was purchased from Santa Cruz Biotechnology. Animals and treatment Fischer 344 female ovariectomized (OVEX) rats of 180–240 g body weight were obtained from Simmonson Laboratory (Gilron, CA). Animals were housed in animal facilities certified by the American Association for the Accreditation of Laboratory Animal Care. Rats were maintained on a 12 h light and 12 h dark cycle, and fed rodent chow meal (Purina Mills, Inc., St. Louis, MO) and given tap water ad libitum. The animal studies were carried out in adherence to the guidelines established in the ‘Guide for the Care and Use of Laboratory Animals’, US Department of Health and Human Resources (NIH 1985). Those animals showing regular 4-day estrous cycles were used for the study. Groups of animals (eight animals per group) were surgically implanted with a 1-cm silastic capsule filled with 17β-estradiol (Sigma Chemical Co., St. Louis, MO). The implantation procedure was identical as described by Burns and Sarkar (32). Animals were treated with E2 for different intervals (e.g. 7.0, 15.0 and 30.0 days) and were killed by rapid decapitation. Pituitaries were excised, and some (three pituitaries/ group) were immediately frozen in liquid nitrogen and stored at 280°C for Western blot analysis. The remainder were fixed in 10% formalin-saline, and processed for paraffin embedding and tissue sectioning. Controls consisted of age and weight-matched OVEX rats receiving the same diet but empty silastic capsule. Histological assessment of tumor angiogenesis in pituitary To identify the histological development of tumor angiogenesis in rat pituitary, immunohistochemical examination for FVIIIRAg was performed. Microvessel quantitation was accomplished according to the methods of Brem et al. (33) and Bosari et al. (34). To determine blood vessel density, FVIIIRAg immunostained sections were first scanned under low power magnification (350) for microvessel ‘hot spot’ (areas with the most dense vascular staining). The microvessel density at 3250 field was then determined by averaging the number of blood vessels in the three most vascular areas (hot spot) of a section. The result was always confirmed by a second, blinded investigator. The blood vessels were classified into three categories according to their sizes (i.e. small, medium and large). Blood vessels ,5 µ were classified as ‘small’, vessels .5 and ,12 µ in size were classified as ‘medium’, .12 µ were considered ‘large’. At least five sections from each pituitary were examined for vascular density assessment. Western immunoblotting analysis Frozen pituitary glands were washed twice with cold 10 mM phosphate buffer saline, pH 7.4 (13PBS) and homogenized in lysis buffer (10 mM Tris, pH 7.4, 1 mM EDTA, 50 mM NaCl, 0.5% Nonidet P-40, 1 mM dithiotreitol, 1 mM phenylmethylsulfonyl fluoride, and 10 µg/ml of leupeptin and aprotinin) for 30 s in ice cold condition. Insoluble material was removed from the homogenate by centrifugation at 14 000 rpm for 30 min at 4°C. Protein concentration was determined by Coomassie blue method (Piearce, Rockford, IL) using bovine serum albumin as a standard. Soluble proteins (50 µg) were separated by electrophoresis in 12% SDS–PAGE gels and then transferred to nitrocellulose papers (BioRad, CA) using transblot apparatus (BioRad). Non-specific binding sites were blocked by incubating the filters in blocking solution (Piearce) for 30 min at room temperature. The filters were then incubated for overnight at 4°C with either rabbit anti-VEGF polyclonal anitbody (2 µg/ml in TBS–0.05% Tween 20) or pre-immune serum which served as a negative control. After washing twice with TBS–0.05% Tween 20, the blots were incubated with HRP conjugated antirabbit IgG (Piearce, Rockford, IL) for 1 h at room temperature. The antigen– antibody complexes were visualized by ECL Cheminufluorescence detection system. Immunohistochemistry Immunohistochemical staining procedure was carried out according to manufacturer’s instructions (Zymed, CA) with few modifications. Briefly, Paraffinembedded 10 µm tissue sections were dewaxed in xylene and rehydrated in 13PBS, through different concentrations of ethanol. To block the endogenous peroxidase activity, slides were incubated in 3% hydrogen peroxide (3% v/v) in methanol for 5 min at room temperature. The slides were then incubated in citrate buffer (Zymed, CA) in a microwave oven at high power for 5 min and allowed to cool for 15 min. After washing the slides in water followed by 13PBS for 5 min, sections were incubated in ready to use tissue blocker for 15 min at room temperature. Tissue blocker was replaced with primary antibodies or preimmune IgG as negative controls and incubated overnight at 4°C. Antibodies used were a rabbit polyclonal antibody at a 1:300 dilution for VEGF (Neomarkers, CA); a rabbit polyclonal antibody at a 1:200 dilution for VEGF-R2

1156

(KDR/Flk-1) (Santa Cruz Biotechnology Inc., CA), and a ready to use polyclonal antibody for FVIIIRAg (Zymed Laboratories Inc., CA). After incubation with primary antibodies, slides were rinsed with 13PBS (335 min), and were incubated for 10 min at room temperature in biotinylated rabbit-anti-goat IgG. After rinsing with PBS (335 min), sections were incubated in peroxidase-conjugated linker for 10 min at room temperature. VEGF/VPF or VEGFR-2 was detected by an incubation with 3,39-diaminobenzidine tetrahydrochloride solution (Zymed Co., CA) for a period of time sufficient to yield dark brown color, usually 5 min. Sections were counter stained with hematoxylin for microscopic examination. Evaluation of VEGF/VPF immunostaining The intensity of VEGF immunostaining was graded on a scale of 1 to 3. 1 represents faint brown immunostained cells or blood vessels; 2 represents moderately strong brown immunostained cells or vessels; and 3 indicates dark brown immunostained cells or vessels. Lectin–histoperoxidase binding assay To identify the endothelial cells involved in angiogenesis in rat pituitaries, an endothelial cell specific marker (35), lectin UEA1-binding study was performed. Lectin-binding assay technique was performed as described by Williams et al. (36) with some modifications. Briefly, paraffin-embedded tissue sections were dewaxed in xylene and rehydrated in 13PBS, through different concentrations of ethanol. Endogenous peroxidase activity was blocked by immersion of slides in 3% hydrogen peroxide in methanol for 5 min. The slides were washed in 13PBS and after blot drying, sections were incubated with a biotin-conjugated lectin solution in 13PBS (12.5 µg/ml) at 37°C for 45 min. After rinsing with 13PBS (335 min), sections were incubated with 3,39-diaminobenzidine tetrahydrochloride solution (Zymed, CA), for a time sufficient to yield a dark brown color, usually 5 min. Sections were counter stained lightly with haematoxylin. Statistical analysis The data were analysed by Student’s t-test and by one way ANOVA with the criterion for statistical significance P 5 0.05.

Results Quantitation of microvessels during the development of estrogen-induced pituitary tumors Examples of FVIIIRAg immunostained positive microvessels of different sizes, and the mean numbers of microvessel density are shown in Figure 1 and Table I, respectively. The distribution patterns of the three sizes of blood vessels were markedly different in the pituitary of E2-treated ovariectomized rats compared with untreated ovariectomized rats. The large-sized blood vessels’ counts increased 17- and 22-fold relative to untreated age-matched controls after 7 and 30 days of E2 exposure, respectively. After 30 days of E2 treatment, smallsized blood vessel density declined by .93% compared to untreated controls with a majority of the surface of the sections occupied by medium and large size blood vessels. Level of VEGF/VPF protein expression in pituitaries during estrogen treatment measured by Western blot To determine the VEGF protein expression levels in rat pituitaries during E2 treatment, Western blot analysis was performed on 50 µg of protein extracted from estrogen-treated and age-matched untreated rat pituitaries, using anti-VEGF antibodies. A representative of Coomassie blue stained gel exhibiting equal amounts of protein concentration per sample, VEGF Western blot and the results are shown in Figure 2. A band of 28–31 kDa rat specific for VEGF protein was detected in both untreated normal and estrogenized pituitaries. However, a dramatic increase of VEGF-specific protein was observed after prolonged estrogen treatment. VEGF protein expression levels exhibited 4.0- and 5.5-fold elevations in pituitary after 7 and 15 days of E2 exposure, respectively. After 15 days of E2 exposure the VEGF expression level was abruptly plateau (data not shown).

Estrogen-initiated tumor angiogenesis

Fig. 2. Western blot analysis of E2 effect on VEGF synthesis in the pituitary of ovarectomized rats during the development of estrogen-induced pituitary tumors. (a) Single representative of Coomassie blue stained gel exhibiting equal amount of protein loaded in each lane; (b) single representative blot showing VEGF/VPF expression in the pituitary of untreated and E2-treated rats and (c) arbitrary values indicate VEGF/VPF protein concentrations in the pituitary of untreated and E2 treated rats. Data displayed as mean 6 SEM in each case. P value was determined by Student’s t-test: *P , 0.05; **P , 0.005 as compared to untreated agematched control rats received no hormone implants. Fig. 1. Examples of sequential changes in size of microvessel localized by immunostaining with factor VIII-related antigen. (a) 7 days E2-treated pituitary section; (b) 15 days E2-treated pituitary section; and (c) 30 days E2-treated pituitary section. Arrow heads indicate small vessels (,5 µ); small arrows indicate medium vessels (.5–,12 µ), and large arrows indicate large vessels (.12 µ). Bar indicates 10 µm.

Immunohistochemistry of VEGF in normal and estrogentreated pituitary of rats Since Western blot analysis provided limited information as to the cell type-specific protein expression in the anterior pituitary, VEGF/VPF immunohistochemistry was performed to examine

cell specific accumulation of VEGF/VPF protein as well as the intensity and density of VEGF positive cells and blood vessels. In untreated OVEX pituitary sections, weak staining signals were detected with antibodies to VEGF in a separate pituitary cell population and surrounding blood vessels (Figure 3b). Strong VEGF immunostaining was observed after E2 treatment (Figure 3c2d), indicating VEGF expression increased in the pituitary of OVEX rats during estrogen treatment. VEGF/VPF protein expression in the untreated rat anterior pituitary sections was primarily restricted within the blood vessels. A few separate cells (0.6% 6 0.3) exhibited weak reactions in the untreated 1157

S.K.Banerjee et al.

Fig. 3. Examples of immunohistochemical localization of VEGF/VPF in OVEX rat pituitary sections during estrogen-induced tumorigenesis. (a) Negative control pituitary section immunostained with pre-immune serum in place of the polyclonal anti-VEGF; (b) untreated control pituitary section exhibiting VEGF negative blood vessels (large arrows) and VEGF positive (weakly) blood vessels (small arrows); (c) 7 days E2-treated pituitary section exhibiting strong VEGF-positive cells (arrows); (d) 7 days E2-treated tissue demonstrating strong VEGF positive blood vessels (arrows). 3400.

Table I. Effect of 17β-estradiol on induction and modulation of blood vessels in the pituitary of rats Agents

OVEX untreated

Estrogen 17β-estradiol (E2)

Treatment [days]

Blood vessels densitya Small

Medium

Large

7 15 30

23.9 6 3.5 21.4 6 2.5 26.1 6 2.7

1.7 6 1.0 2.4 6 0.8 4.6 6 2.0

0.2 6 1.2 0.0 0.3 6 0.2

7 15 30

12.2 6 1.0** 14.1 6 3.1** 1.1 6 0.2**

5.5 6 1.9* 6.0 6 1.0* 4.2 6 1.2*

2.9 6 0.3** 4.0 6 0.2 6.7 6 1.0**

are displayed as mean numbr of VIIIRAg1 blood vessels 6 SE. *P , 0.05 compared with the untreated group; **P , 0.005 compared with the untreated group. aResults

control sections (Table II). Strong VEGF expressions were observed in these cells after 7 days of E2 exposure (Figure 3c; Table II). The number of VEGF/VPF positive cells increased 9.5-fold relative to age-matched controls after 7 days of E2 exposure (Table II). The signals as well as the number of VEGF/ VPF immunostained positive separate cells declined sharply after 15 days of E2 exposure (Table II), although the data shows that the number of VEGF/VPF positive cells in the E2-treated group was significantly higher than controls. None of this cell 1158

population exhibited a positive staining reaction with antibodies to VEGF after 30 days of E2 treatment (data not shown). Moreover, the intensity of VEGF/VPF protein immunostained in blood vessels and the number of VEGF immunostained positive blood vessels were significantly increased in E2-treated groups (Table II, and Figure 3d). The density of VEGF/VPF positive blood vessels increased to 3.0- and 4.0-fold after 7 and 15 days of E2 treatment, respectively (Table II). No specific staining was noted when sections were immunostained with pre-immune serum (Figure 3a). Immunohistochemistry of VEGFR2 (KDR/Flk-1) in untreated and estrogen-treated anterior pituitary Immunohistochemical analysis was performed to determine the cell specific expression and density of VEGF receptor (VEGFR-2) during the development of estrogen-induced pituitary tumors. Results indicate that VEGFR-2 protein synthesis was restricted to the blood vessels in both untreated and E2 treated pituitary sections (Figure 4). The density of the immunostained positive blood vessels increased 3.5- and 6.7fold after 7 and 15 days of E2 exposure, respectively. It was noted that the rate of increase in density of VEGF R-2 positive vessels was markedly accelerated during chronic exposure of E2 (Figure 5). Unlike VEGF, VEGFR-2 protein expression was not detected in the separate cell population. This finding suggests that the separate cell populations that were immunoreacted with VEGF antibodies are non-endothelial cells. No

Estrogen-initiated tumor angiogenesis

Fig. 4. Examples of immunohistochemical localization of VEGFR-2 (KDR/Flk-1) in ovarectomized rat pituitary sections during estrogen-induced tumorigenesis. (a) Negative control pituitary section immunostained with pre-immune serum in place of the polyclonal anti-KDR/Flk-1; (b) untreated control pituitary section exhibiting VEGFR-2 negative blood vessels; (c) 7 days E2-treated demonstrating strong VEGFR-2 positive blood vessels; and (d) 15 days E2-treated tissue demonstrating strong VEGFR-2 positive blood vessels. 3400.

Table II. Intensity of VEGF immunostaining during estrogen-induced pituitary tumorigenesis in rats Duration of treatment (days)

Intensity of VEGF staining (% mean 6 SEM)a Separate pituitary cells

Untreated 7 15 E2-treated 7 15

Blood vessels

1

2

3

1

0.65 6 0.3 0.54 6 0.2

0 0

0 0

1.1 6 0.8* 2.0 6 0.5*

4.3 6 0.7 0.7 6 0.3

0.8 6 0.3 0

2

3

8.0 6 1.0 7.6 6 0.7

0.8 6 0.3 1.0 60.4

17.2 6 1.0** 19.8 6 1.3**

8.4 6 1.1** 10.6 6 1.2**

0 0 2.4 6 0.5 5.2 6 0.4

a1,

faint brown stain; 2, moderately strong brown stain; 3, dark brown stain. *P , 0.05 compared with the untreated group; **P , 0.005 compared with the untreated group.

specific immunostained was noted when sections were immunostained with pre-immune serum (Figure 4). Identification of cell-type in the estrogen-induced pituitary tumors To determine the cell type in the estrogen treated pituitary sections that immunoreacted with VEGF/VPF antibodies during estrogen exposure, endothelial cell specific lectin binding assay and FVIIIRAg immunostaining was performed in the adjacent tissue sections. Result demonstrated that the lectin binding and

FVIIIRAg immunoreaction were detected in the blood vessels (Table III). Endothelial specific lectin and FVIIIRAg was not reacted with VEGF positive separate cell populations (Table III). Discussion Previous studies from different laboratories have suggested that tumor angiogenesis is indispensable for the induction and growth of estrogen-induced prolactin-secreting pituitary tumors in Fischer 344 rats (4–6). The present histological studies corrobor1159

S.K.Banerjee et al.

Fig. 5. Immunohistochemical analysis of E2 effects on VEGFR-2 (KDR/ Flk-1) expression in the pituitary of ovariectomized rats during the development of estrogen-induced pituitary tumors. E2-treated and untreated rats were killed at indicated intervals. The frequency of VEGFR-2 immunopositive microvessels in the pituitary sections was performed by VEGFR-2 (KDR/Flk-1) immunohistochemistry. The frequency was calculated by counting the number of labeled microvessels divided by the total number of vessels times one hundred (%). The frequency of VEGFR-2 positive microvessels in each pituitary was determined from five randomly selected sections. At least five rats were used for each time point. Data is displayed as mean 6 SEM in each case. P value was determined by Student’s t-test: *P , 0.05; **P , 0.005 as compared to untreated age-matched control rats received no hormone implants.

Table III. Identification of endothelial cells in pituitary using the factor VIII-related antigen, lectin, UEA1 and VEGFR-2 Antibody/lectin

VEGF positive cellsa

VEGF positive blood vesselsa

Factor VIII-related antigen Lectin VEGFR-2

2 2 2

1 1 1

a2,

no immunoreaction with the specific antibodies; and 1, immunoreacted with specific antibodies.

ated earlier observations and extended the findings by demonstrating the induction of neovascularization during the early stages of estrogen-induced tumorigenesis. Furthermore, the results of the quantitative analysis of different sizes of microvessels suggest that estrogen not only induces neovascularization in the anterior pituitary, it also stimulates the growth and enlargement of blood vessels, to direct and maintain blood flow and tissue perfusion, which eventually might be required for the rapid growth of pituitary lactotrops. It is uncertain how estrogen regulates the tumor angiogenic switch in the anterior pituitary of rats during estrogen carcinogenesis, and none of the previous studies have uncovered the potential molecular events regulating tumor angiogenesis in rat pituitaries. To elucidate the possible mechanism of estrogen induced tumor angiogenesis in the pituitary of female rats, VEGF/VPF and 1160

VEGFR-2 protein expression were examined in both untreated and E2-treated pituitaries using Western blot and immunohistochemistry. The studies demonstrated that VEGF and its receptor, VEGFR-2 protein expression were enhanced significantly in the pituitary after 7 days of estrogen treatment, and the level of expression markedly accelerated during prolonged exposure. The over expression of VEGF/VPF and VEGF receptors correlates with endothelial cell proliferation, formation of new blood vessels and tumor angiogenesis (37). Thus, the elevation of VEGF expression and induction of neovascularization and enlargement of blood vessels in the pituitary during estrogen treatment raises the possibility that VEGF along with its receptor-2, may play a critical role in turning the angiogenic switch on in the anterior pituitary of rats, during the early stage of estrogen-induced tumorigenesis. The cell type specific distribution of VEGF and VEGFR-2 was determined by FVIIIRAg and lectin UEA1 immunoperoxidase staining of untreated and E2-treated adjacent pituitary sections (Table III). FVIIIRAg and lectin UEA1 employed in this study have been shown to bind to new or existing endothelial cells (35,38). The present studies indicated that FVIIIRAg and UEA1 immunoreacted with microvascular endothelial cells, but not with the subsets of VEGF immunopositive pituitary cells, suggesting that both endothelial and nonendothelial pituitary cells were actively involved in synthesizing VEGF after 7 days of estrogen treatment. The expression of VEGF protein in different cell types, as observed herein, is consistent with previous findings of VEGF mRNA expression in human and mouse epithelial cells (31,39,40). The intriguing part of these observations was that VEGF expression declined in subsets of non-endothelial cells after 15 days of E2 exposure. In contrast, VEGF expression was up regulated in the microvessel endothelial cells during chronic exposure of estrogen. The functional significance of the induction of this growth factor expression in the non-endothelial pituitary cells is uncertain. Since these cells are adjacent to the blood vessels, a paracrine relationship between the two populations of cells may exist. The over expression of VEGF in the anterior pituitary may overrule the signals of negative regulator(s) (41,42) thereby turning the angiogenic switch on in the endothelial cells. VEGFR-2 signals were localized exclusively to microvascular endothelial cells during the early phase of estrogen-induced tumorigenesis. In particular, the marked elevation of VEGFR-2 expression in the microvascular endothelial cells was observed after 15 days of E2 exposure. This study was consistent with earlier reports (26) and suggest that over production of VEGF in the estrogen treated anterior pituitary, may be activating VEGFR-2 in microvascular endothelial cells required for endothelial differentiation (37). Finally, since VEGFR-2 was selectively expressed in the microvascular endothelial cells (26), this finding is an additional indication that VEGF positive separate anterior pituitary cell populations are non-endothelial in nature. However, more studies are required for further confirmation. In summary, our results systematically analysed the effect of estrogen on VEGF and VEGFR-2 protein synthesis during the development of estrogen-induced tumor angiogenesis in F344 rat pituitary. The studies suggested that VEGF over expression in the anterior pituitary may be associated with the early events in the development of estrogen-induced tumor angiogenesis that, in turn, induced the growth of prolactin-secreting pituitary tumors. Additionally, the over expression of VEGFR2 protein in the blood vessels, indicates that VEGFR-2

Estrogen-initiated tumor angiogenesis

also plays an important role in the regulation of neovascularization associated with the development of estrogen-induced pituitary tumors in F344 rats. Acknowledgements This work was supported by the MidWest Biomedical Research Foundation, Kansas City, MO to SKB and NIH grant (CA 56056) to DKS.

References 1. Henderson,B.E., Ross,R. and Bernstein,L. (1988) Estrogen as a cause of human cancer. Cancer Res., 48, 246–253. 2. Barrett,J.C. (1992) Molecular mechanisms of hormonal carcinogenesis. In Li,J.J., Nandi,S. and Li,S.A. (eds) Hormonal Carcinogenesis. SpringerVerlag. New York, vol. 1, pp. 159–163. 3. Furth,J. and Clifton,K.H. (1966) Experimental pituitary tumors. In Harris,G.W. and Donovan,B.T. (eds) The Pituitary Gland. Butterworths, London, pp. 460–497. 4. Elias,K.A. and Weiner,R.I. (1984) Direct arterial vascularization of estrogeninduced prolactin-secreting anterior pituitary tumors. Proc. Natl Acad. Sci. USA, 81, 4549–4553. 5. Elias,K.A. and Weiner,R.I. (1987) Inhibition of estrogen-induced anterior pituitary enlargement and arteriogenesis by bromocriptine in Fisher 344 rats. Endocrinology, 120, 617–621. 6. Takechi,A., Uozumi,T., Kawamoto,K., Ito,A., Kurisu,K. and Sudo,K. (1994) Inhibitory effect of TNP-470, a new anti-angiogenic agent, on the estrogen induced pituitary tumors. Anticancer Res., 14, 157–162. 7. Cockerill,G.W., Gamble,J.R. and Vadas,M.A. (1995) Angiogenesis: model and modulators. Int. Rev. Cytol., 159, 113–159. 8. Klagsburn,M. and D’Amore,P.A. (1991) Regulators of angiogenesis. Ann. Rev. Physiol., 53, 217–239. 9. Berkman,R.A., Merrill,M.J., Reinhold,W.C., Monacci,W.T., Saxena,A., Clark,W.C., Robertson,J.T., Ali,I.U. and Oldfield,E.H. (1993) Expression of vascular permeability factor/vascular endothelial growth factor gene central nervous system neoplasms. J. Clin. Invest., 91, 153–159. 10. Zagzag,D. (1995) Angiogeneic growth factors in neural embryogenesis and neoplasia Am. J. Pathol., 146, 293–309. 11. Takano,S., Yoshii,Y., Kondo,S., Suzuki,H., Maruno,T., Shirai,S. and Nose,T. (1996) Concentration of vascular endothelial growth factor in the serum and tumor tissue of brain tumor patients. Cancer Res., 56, 2185–2190. 12. Takahashi,Y., Kitadai,Y., Bucana,C.D., Cleary,K.R. and Ellis,L.M. (1995) Expression of vascular endothelial growth factor and its receptor, KDR, correlates with vascularity, metastasis, and proliferation of human colon cancer. Cancer Res., 55, 3964–3968. 13. Brown,L.F.. Berse,B., Jackman,R.W., Tognazzi,K., Manseau,E.J., Senger,D.R. and Dvorak,H.F. (1993) Expression of vascular permeability factor (vascular endothelial growth factor) and its receptors in adenocarcinomas of the gastrointestinal tract. Cancer Res., 53, 4727–4735. 14. Olson,T.A., Mohanraj,D., Carson,L.F. and Ramkrishnan,S. (1994) Vascular permeability factor gene expression in normal and neoplastic human ovaries. Cancer Res., 54, 276–280. 15. Yoshiji,H., Gomez,D.E., Shibuya,M. and Thorgeirsson,U.P. (1996) Expression of vascular endothelial growth factor, its receptor, and other angiogenic factors in human breast cancer. Cancer Res., 56, 2013–2016. 16. Kim,K.J., Li,B., Winer,J., Armanini,M., Gillett,N., Phillips,H.S. and Ferrara,N. (1993) Inhibition of vascular endothelial growth factor-induced angiogenesis suppresses tumor growth in vivo. Nature (Lond.), 362, 841–844. 17. Asano,M., Yukita,A., Matsumoto,T., Kondo,S. and Suzuki,H. (1995) Inhibition of tumor growth and metastasis by an immunoneutralizing monoclonal antibody to human vascular endothelial growth factor/vascular permeability factor121. Cancer Res., 55, 5296–5301. 18. Saleh,M., Stacker,S.A. and Wilks,A.F. (1996) Inhibition of growth of C6 glioma cells in vivo by expression of antisense vascular endothelial growth factor sequence. Cancer Res., 56, 393–401. 19. Ferrara,N., Carver-Moore,K., Chen,H., Dowd,M., Lu,L., O’Shea,S., PowellBraxton,L., Hillan,K.J. and Moore,M.W. (1996) Heterozygous embryonic lethality induced by targeted inactivation of the VEGF gene. Nature, 380, 439–342. 20. Ferrara,N., Winer,J., Burton,T., Rowland,A., Siegel,M., Phillips,H.S., Terrell,T., Keller,G.A. and Levinson,A.D. (1993) Expression of vascular endothelial growth factor does not promote transformation but confers a growth advantage in vivo to chinese hamster ovary cells. J. Clin. Invest., 91, 160–170. 21. Shibuya,M., Yamaguchi,S., Yamane,A., Ikeda,T., Tojo,A., Matsushime,H. and Sato,M. (1990) Nucleotide sequence and expression of a novel human

receptor type tyrosine kinase (flt) closely related to the fms family. Oncogene, 5, 519–524. 22. de Vries,C., Escobedo,J.A., Ueno,H., Houck Ferrara,N. and Williams,L.T. (1992) The fms-like tyrosine kinase, a receptor for cellular endothelial growth factor. Science, 255, 989–991. 23. Terman,B.I., Dougher-Vermazen,M., Carrion,M.E., Dimitrov,D., Armellino, D.C., Gospodarowicz,D. and Bohlen,P. (1992) Identification of KDR tyrosine kinase as a receptor for vascular endothelial cell growth factor. Biochem. Biophys. Res. Commun., 187, 1579–1586. 24. Mathews,W., Jordan,C.T., Gavin,M., Jenkins,N.A., Copeland,N.G. and Lemischka,I.R. (1991) A receptor tyrosine kinase cDNA isolated from a population of enriched primitive hematopoietic cells and exhibiting close genetic linkage to c-kit. Proc. Natl Acad. Sci. USA, 88, 9026–9030. 25. Quinn,T.P., Peters,K.G., DeVries,C., Ferrara,N. and Williams,L.T. (1993) Fetal liver kinase 1 is a receptor for vascular endothelial growth factor and is selectively expressed in vascular endothelium. Proc. Natl Acad. Sci. USA, 90, 7533–7537. 26. Millauer,B., Wizigmann-Voos,S., Martinez,R., Moller,N.P.H., Risau,W. and Ullrich,A. (1993) High affinity VEGF binding and developmental expression suggest Flk-1 as a major regulator of vasculogenesis and angiogenesis. Cell, 72, 835–846. 27. Peters,K.G., De-Vries,C. and Williams,L.T. (1993) Vascular endothelial growth factor receptor expression during embryogenesis and tissue repair suggests a role in endothelial differentiation and blood vessel growth. Proc. Natl Acad. Sci. USA, 90, 8915–8919. 28. Waltenberger,J., Claesson-Welsh,L., Siegbahn,A., Shibuya,M. and Heldin, C.-H. (1994) Different signal transduction properties of KDR and Flt1, two receptors for vascular endothelial growth factor. J. Biol. Chem., 269, 26988–26995. 29. Millauer,B., Shawver,L., Plate,K., Risau,W. and Ullrich,A. (1994) Glioblastoma growth inhibited in vivo by a dominant-negative Flk-1 mutant. Nature (Lond.), 367, 576–579. 30. Kolch,W., Martiny-Baron,G., Kieser,A. and Marme,D. (1995) Regulation of the expression of the VEGF/VPS and its receptors: role in tumor angiogenesis. Breast Cancer Res. Treat., 36, 139–155. 31. Cullinan Bove,K. and Koos,R.D. (1993) Vascular endothelial growth factor/ vascular permeability factor expression in rat uterus: rapid stimulation by estrogen correlates with estrogen-induced increases in uterine capillary permeability and growth. Endocrinology, 133, 829–837. 32. Burns,G. and Sarkar,D.K. (1993) Transforming growth factor-beta-1-like immunoreactivity in the pituitary gland on the rat: effect of estrogen. Endocrinology, 133, 1444–1449. 33. Berm,S., Cotran,S.S. and Folkman,J. (1972) Tumor angiogenesis: a quantitative method for histological grading. J. Natl Cancer Inst., 48, 347– 356. 34. Bosari,S., Lee,A.K., DeLellis,R.A., Wiley,B.D., Heatley,G.J. and Silverman,M.L. (1992) Microvessel quantitation and prognosis in invasive breast carcinoma. Hum. Pathol., 23, 755–761. 35. Petrovic,L.M., Burroughs,A. and Scheuer,P.J. (1989) Hepatic sinusoidal endothelium: Ulex lectin binding. Histopathology, 14, 233–243. 36. Williams,R.J., Robertson,D. and Davies,A.J. (1989) Identification of vascular endothelial cells in murine omentum using lectin. Dolichos biflorus agglutinin: possible applications in the study of angiogenesis. Histochem. J., 21, 271–278. 37. Risau,W. (1995) Differentiation of endothelium FASEB J., 9, 926–933. 38. Samoto,K., Ikezaki,K., Yokoyama,N. and Fukui,M. (1994) P-glycoprotein expression in brain capillary endothelial cells after focal ischaemia in rat. Neurol. Res., 16, 217–223. 39. Sharkey,A.M., Charnock-Jones,D.S., Boocock,C.A., Brown,K.D. and Smith,S.K. (1993) Expression of mRNA for vascular endothelial growth factor in human placenta. Reprod. Fertil., 99, 609–615. 40. Charnock-Jones,D.S., Sharkey,A.M., Rajput-Williams,J., Burch,D., Schofield,J.P., Fountain,S.A., Boocock,C.A. and Smith,S.K. (1993) Identification and localization of alternately spliced mRNAs for vascular endothelial growth factor in human uterus and estrogen regulation in endometrial carcinoma cell lines. Biol. Reprod., 48, 1120–1128. 41. Hanahan,D. and Folkman,J. (1996) Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell, 86, 353–364. 42. Liotta,L.A., Steeg,P.S. and Stetler-Stevenson,W.G. (1991) Cancer metastasis and angiogenesis: an imbalance of positive and negative regulation. Cell, 64, 327–336. Received on October 11, 1996; accepted on February 13, 1997

1161