Download The Edpm5 locus prevents the `angiogenic switch

Carcinogenesis vol.25 no.10 pp.1829--1838, 2004
doi:10.1093/carcin/bgh192
The Edpm5 locus prevents the ‘angiogenic switch’ in an estrogen-induced rat
pituitary tumor
Jyotsna Pandey, Anas Bannout and Douglas L.Wendell1
Department of Biological Sciences, Oakland University, Rochester,
MI 48309, USA
1
To whom correspondence should be addressed
Email: [email protected]
Edpm5 is one member of a group of quantitative trait loci
that are responsible for the difference in susceptibility to
estrogen-induced prolactinoma between the Fischer 344
(F344) and Brown Norway (BN) strains. Upon chronic
estrogen treatment F344 rats develop large, hemorrhagic
and invasive pituitary tumors, which exhibit both tumor
angiogenesis and neoplasia. In contrast, BN rats do not
develop a tumor despite an estrogen-induced increase in
lactotroph density. To investigate the role of Edpm5 in the
development of these tumors, we have generated a novel
congenic rat strain F344.BN-Edpm5BN by introgressing the
segment of rat chromosome bearing Edpm5 from BN into
the F344 strain background. Phenotypic differences
between F344 and F344.BN-Edpm5BN must be due to a
gene(s) within the chromosomal interval encompassing
Edpm5. Through use of these strains, we find that Edpm5
specifically regulates the switch to angiogenic phenotype,
independent of neoplasia. The F344.BN-Edpm5BN rats
developed tumors, which exhibited significant growth,
7-fold greater mass than the pituitary of untreated rats, and
neoplasia indistinguishable from that of the F344 strain.
However, the F344.BN-Edpm5BN rat tumor had a nonangiogenic phenotype. After chronic estrogen treatment,
there was no increase in microvessel count over untreated
controls in F344.BN-Edpm5BN tumors, whereas F344 rat
tumors showed a significant increase (P 5 0.0005). The
ultrastructural morphology of the pituitary blood vessels
also did not show significant angiogenesis associated
changes in F344.BN-Edpm5BN rat pituitary tumors. In contrast the parental strain F344 had pronounced angiogenic
activity. The F344.BN-Edpm5BN strain also fails to express
VEGF at the high levels seen in the F344 rat pituitary after
estrogen treatment. Hence at least one gene that has a large
impact, directly or indirectly, on the switch to angiogenic
phenotype must reside within the chromosomal interval
that is the Edpm5 quantitative trait locus.
Introduction
The rat pituitary is valuable as an experimental system for
tumor biology because there is great variation between rat
strains (i.e. genetic variation) in many significant tumor phenotypes. Chronic estrogen treatment of rats of the strain
Abbreviations: BM, basement membrane; EC, endothelial cells; GS,
Griffonia simplicifolia; MVC, microvessel count; PRL, prolactin; QTL,
quantitative trail locus; RBC, red blood cells.
Carcinogenesis vol.25 no.10 # Oxford University Press 2004; all rights reserved.
Fischer 344 (F344) induces large tumors, which consist primarily of lactotrophs (1,2). In contrast, tumor-resistant strains
like Holtzman, Sprague--Dawley and Brown Norway (BN), are
able to restrain estrogen-induced growth and do not form
pituitary tumors (1,4--6). Among the tumor phenotypes that
are induced by chronic estrogen treatment in the pituitary of
F344 rats are uncontrolled cell proliferation (4,7), neoplastic
transformation (3,8), tumor angiogenesis (9--11) and (after
prolonged treatment) invasion of surrounding tissue (12). Not
all investigators who have studied the estrogen-induced pituitary tumors of the F344 rat have included a tumor-resistant
strain in their studies, but from those who have, we know that
the processes of cell proliferation (4) and angiogenesis (2,11)
are controlled in tumor-resistant strains so that chronic estrogen treatment does not lead to either increased cell proliferation or increased angiogenesis in the tumor-resistant strains.
After chronic estrogen treatment, the pituitary of F344 rats
exhibit ‘tumor angiogenesis’. Tumor angiogenesis, unlike normal physiological angiogenesis, has features of uncontrolled
angiogenic activity and chaotic vessel architecture (13). Parameters of tumor angiogenesis that have been reported in the
pituitary of F344 rats in response to estrogen treatment are
increased microvessel count (MVC) (10,11,14), loss of integrity and extensive reorganization of basement membrane (BM)
(2,3,11), a torturous and disorganized pattern of vasculature
(11,15--17), and loss of vessel wall integrity and the presence
of vessels unlined by capillary endothelium (2,3,11). In contrast, these features are all much less severe or non-existent in
the pituitary of estrogen-treated BN rats (11). Angiogenesis is
a normal biological process, which is under tight control in
normal tissues (18,19). A paradigm of modern tumor biology
is the ‘angiogenic switch’, namely that tumor angiogenesis is
controlled by a balance of many positive and negative regulatory factors and an imbalance between them is thought to cause
a change to an angiogenic phenotype in tumors (20). Thus, in
comparing the angiogenic phenotype of F344 and BN after
chronic estrogen treatment, we see heritable variation in the
angiogenic switch and we take the view that the BN strain is
proficient in controlling the angiogenic switch while the
F344 strain has mutations that render it unable to control
the angiogenic switch in the pituitary after chronic estrogen
treatment (11).
It should be noted that there is a significant difference
between the estrogen-induced pituitary tumor of the F344 rat
and human prolactinomas. It is firmly established that solid
tumors are dependent on angiogenesis (13,19) and induction of
angiogenesis has been studied as a pre-neoplastic marker for
solid tumors in many tissues (19,21--23). Parameters such as
increased intra-tumor microvessel density are often used to
reflect this (24), though there is some debate as to the appropriate method for counting and if it is the correct parameter for
all tumor types (25). While the F344 rat pituitary clearly shows
increased angiogenic activity and higher vascular counts upon
estrogen treatment (9--11,14) typical of most human solid
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J.Pandey, A.Bannout and D.L.Wendell
tumors, human pituitary tumors do not exhibit increased vascularization and in fact tend to have lower vascular counts than
the normal pituitary gland (26). Thus, in this report we do not
wish to represent the pituitary of the estrogen-treated F344 rat
as a ‘model prolactinoma’, i.e. an animal model that replicates
the symptoms of the specific human disease of prolactinoma.
Instead, we view the angiogenic activity of the F344 rat pituitary tumor as being relevant to tumor angiogenesis in general
and consistent with the phenomena seen in a broad range of
solid tumors (13,19,24,27).
The difference between BN and F344 in their ability to
control estrogen-induced tumor development has been found
to be due to variation in at least six genes, which were identified by quantitative trail locus (QTL) mapping (28,29). Further
mapping studies indicated that the BN allele of one of these
QTL, Edpm5, is associated with a lower level of total pituitary
hemoglobin (29), MMP-9 (30) and VEGF (31). This suggests
that Edpm5 could be involved in regulating tumor angiogenesis, as both VEGF (32,33) and MMP-9 (34,35) are known to
be involved in angiogenesis.
To delineate the effects of BN allele of the Edpm5 locus on
pituitary tumor formation and tumor angiogenesis consequent
to chronic estrogen stimulation, we have developed a new rat
strain, the F344.BN-Edpm5BN congenic strain, by introgressing the segment of rat Chr5 that contains the Edpm5 QTL
from the BN strain into the F344 strain. Thus, by employing
the F344 and F344.BN-Edpm5BN strains in studies of the
estrogen-induced pituitary tumor, we can determine the effect
of Edpm5 independent of other Edpm QTL. We report here our
findings on the effect of BN allele of the Edpm5 QTL on tumor
angiogenesis, which were determined by analysis of the differences in tumor morphology and vasculature after chronic
estrogen treatment of the F344 and F344.BN-Edpm5BN strains.
We find that the BN allele of Edpm5 locus prevents the
‘angiogenic switch’, which is seen in the estrogen-induced
pituitary tumor in F344 rat strain. Furthermore, the BN allele
of Edpm5 can block the angiogenic switch despite significant
increase in mass and neoplastic transformation.
Materials and methods
Construction of the F344.BN-Edpm5BN congenic line
To investigate the contribution of Edpm5 to the control of estrogen-dependent
growth, we constructed a congenic strain in which the segment of rat Chr5
containing the Edpm5 QTL from BN was introgressed into the F344 strain. We
have introgressed a segment of rat Chr5 because, at present, Edpm5 is not
localized to discrete point, but to a chromosomal segment as is typical for QTL
(36). The strain was bred by what is now the standard ‘speed congenic’ strategy
of recurrent backcrossing with selection for donor alleles of microsatellite
markers across the QTL and against donor alleles on all other chromosomes
(37). Rat microsatellite markers were amplified by PCR from DNA extracted
from tail biopsies and resolved by gel electrophoresis as described previously
(31). F344/NHsd (F344) and BN/SsNHsd (BN) were crossed and F1 siblings
were intercrossed. The F2 females that were selected were homozygous for BN
alleles of microsatellite markers in and around Edpm5. (This F2 step is not
typical of congenic line breeding, but was done as part of a different experiment.) Selected F2 females were backcrossed to F344 males and their male
progeny were backcrossed to F344 females. At each backcross generation,
male rats were selected that were carrying a BN allele of markers including and
between D5Mgh5 and D5Mit7. This interval encompasses the 2-LOD support
interval of Edpm5 (28). Of the selected males, animals were further selected for
F344 alleles of microsatellites on other chromosomes. Chromosomes with
known or suggested Edpm QTL (Chr 2, 3, 9 and 14) were intensively monitored with markers spaced at least 20 cM through their QTL interval. All other
chromosomes were monitored with two to four markers (more markers on the
larger chromosomes). This backcrossing with selection was repeated for a
total of four backcross generations. The resulting congenic strain, named
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Fig. 1. Extent of rat Chr5 from the BN strain introgressed into the F344
strain to form the F344.BN-Edpm5BN congenic line. For each microsatellite
marker, a black box indicates that the F344.BN-Edpm5BN congenic line
is homozygous for BN allele of the marker and a white box indicates that the
line was still segregating for F344 and BN alleles in the generation of
rats used in this study. Genetic map distances between markers are as
reported previously (31). All microsatellite markers tested on all
other chromosomes are homozygous for F344 alleles.
F344.BN-Edpm5BN in accordance with the guidelines of the International
Rat Genome Nomenclature Committee (http://rgd.mcw.edu/nomen/rules-fornomen.shtml), is homozygous for BN alleles of microsatellite markers over the
majority of rat Chr5 (Figure 1).
Animals and estrogen treatment
Female rats of three strains, F344, BN and F344.BN-Edpm5BN were used in the
study. Weanling female rats of F344 and BN rat strains were purchased from
Harlan--Sprague--Dawley (Indianapolis, IN) and F344.BN-Edpm5BN were bred
in our colony. For chronic estrogen treatment, 21-day-old rats were given 5 mg
of DES (Sigma, St Louis, MO), in Silastic tubing, implanted subcutaneously as
described previously (38) to induce the pituitary tumor. Animals were killed
10 weeks after receiving the implant (5). DES is used instead of natural estrogen,
such as 17-b-estradiol, because DES has a longer half-life in the body (2). Age
matched rats of each strain without implant were taken as untreated controls.
Animals were housed under controlled climate, 12-h light/dark cycle, with
standard chow and water ad libitum. Oakland University’s Institutional Animal
Care and Use Committee approved all procedures involving live animals.
Tissue sampling and preparation for microscopy
After 10 weeks of estrogen treatment the rats were decapitated and their
pituitaries were collected and weighed. Mass data and notation of gross
morphology were collected on 20 estrogen treated and six untreated rats of
each strain, and tissue from a subset of animals was used for microscopy.
Three pituitaries each of the treated and control rats of all strains were fixed
immediately in 2.5% glutaraldehyde and processed by embedding in epoxyaraldite for electron microscopy (EM) as described earlier (11). Selected
blocks of treated and control pituitary of each strain were sectioned at 1 mm
and examined by light microscopy (LM) after staining with toluidine blue.
Ultra thin sections stained with both uranyl acetate and lead citrate were then
examined for blood vessel ultrastructure using a Phillips electron microscope.
Three pituitaries of control rats and six of the treated rats of each strain were
fixed in 4% formaldehyde for routine processing and embedding for paraffin
sectioning. Paraffin-embedded tissue from control and treated animals were
sectioned at 6-mm thickness and used for routine hematoxylin and eosin
(H&E) stain and immunohistochemistry.
Reticulin stain
Only light microscopy was done for the histopathological classification of the
tumors. EM appears to be of little or no value in the cytological diagnosis of
pituitary prolactinoma as most cells fit the description of ‘normal’ cell type
(39). Reticulin stain was done to differentiate hyperplasia from adenoma.
Edpm5 prevents the angiogenic switch
Gomori’s reticulin stain was used according to the method described by
Culling et al. (40), as the key to distinguishing adenohypophyseal hyperplasia
lies in the breakdown of reticulin network. Hyperplasia is characterized by
expanded acini with an intact reticulin fiber network.
Immunohistochemical detection of prolactin (PRL) and Griffonia simplicifolia
(GS) lectin
The immunohistochemical staining procedure was performed according to the
technique outlined by Shull et al. (41) with modifications as described previously (11) and the additional modification of using Vector Nova-red (Vector
Laboratories, Burlingame, CA) as the chromogenic substrate. Anti-rat PRL
antiserum (National Hormone and Pituitary Program, NIDDK, NIH lot number
AFP425-10-91) was used at 1:32 000 dilution and 20 mg/ml of biotinylated GS
lectin (Vector Laboratories) was used.
Quantification of lactotroph density
Quantification of lactotroph density was carried out on anti-PRL stained
sections, four separate random fields were chosen as starting points and color
images (600) were captured using a digital camera, taking care not to overlap
fields. The captured images were printed out at 1440 d.p.i. on standard 11 8
in, high quality photo paper. Total number of immuno-reactive lactotrophs
versus non-reactive pituicytes was counted, and percentage of lactotrophs was
calculated per sample. The results were averaged for each genotype.
Analysis of MVC and density
Tumor vascularity measurement was performed in order to provide a histological assessment of tumor angiogenesis. For this, the method of choice is
counting MVC in areas of neoangiogenesis or ‘vascular hotspots’ (42,43).
Sections probed with GS lectin, as described above, were used. MVC was
calculated manually as outlined by Weidner et al. (42), with few modifications
(11). Two areas of highest microvessel density containing the highest number
of capillaries (microvessels) per area (i.e. areas of intense vascularity) were
identified by light microscopy, scanning at low power (50 and 100) and
used for microvessel counts by consensus (J.P. and A.B. for Edpm5 Congenic
rats). This was done because, due to the clonal origin of angiogenesis, angiogenic activity is confined to these neovascular ‘hot spots’ (42). Large hemorrhagic regions (lakes) and regions with dilated and congested vessels and
necrosis were excluded. A single countable microvessel was defined as any
endothelial cell or group of cells that was clearly separate from other vessels
and surrounding pituitary tissue by identifying GS lectin positivity, without the
necessity of having a vessel lumen or red blood cells (RBC) within the lumen.
Individual microvessels were counted at 200 (20 objective, 10 eyepiece
lens, 0.7386 mm2 /field), within each hotspot identified. Data were averaged to
give the MVC for each sample.
Western blot detection and quantification of VEGF
Pituitary tissue used for western blots was frozen immediately after dissection
and stored at 80 C. VEGF was detected in western blots as described
previously (31). The primary antibody was the anti-VEGF antibody A-20
from Santa Cruz Biotechnology (catalog # sc-152, Santa Cruz Biotechnology,
Santa Cruz, CA) and was used at 1/1000 dilution. Actin was used as an internal
control for protein loading and was detected with an anti-actin antibody from
Sigma (catalog # A2066, Sigma-Aldrich, St Louis, MO). Secondary antibody
detection was performed using the ECL Plus reagent kit and a Molecular
Dynamics STORM fluorimager (Amersham Biosciences, Sunnyvale, CA).
Band intensities were measured using Molecular Dynamics ImageQuant
software (Amersham Biosciences). An index of VEGF level was computed
as the ratio of VEGF band intensity to the intensity of the actin band within the
same lane.
Statistical analysis
Significance testing was done by t-test (pairwise comparisons of MVC and
lactotroph densities) and Mann--Whitney Rank sum test (pituitary mass) using
SigmaStat, version 2.0 (SPSS, Chicago, IL).
Results
Effect of BN allele of Edpm5 QTL on estrogen-induced
pituitary tumor growth
Upon chronic estrogen treatment, the pituitaries of F344.BNEdpm5BN rats undergo significant pituitary tumor growth, with
mass increasing on average 7-fold over untreated rats. However, the tumors they develop are smaller than those developed
by estrogen-treated F344 rats (Figure 2A).
As in the case of the F344 strain, the tumor of the estrogentreated F344.BN-Edpm5BN rat is a prolactinoma, i.e. after
extensive growth, the tissue consists primarily of prolactin
positive cells (Figure 2B--D). Given that the F344.BNEdpm5BN tumor is seven times normal mass and consists of
86 9% lactotrophs, its growth must involve lactotroph proliferation. Consistent with what we have reported previously
(11), our lactotroph density values, for both treated and
untreated rats of all strains, are significantly higher than
those reported by others (41,44,45). We have no explanation
for this, but immunohistochemistry images are provided in
Figure 2 to support our counts.
As reported previously (5), BN does not develop a tumor
after chronic estrogen treatment and its pituitary has normal
morphology, while treated F344 pituitaries are reddish-brown
in color, symmetrical, large, with prominent areas of hemorrhage and a stretched glistening capsule. In estrogen treated
rats of both F344 and F344.BN-Edpm5BN a fraction of pituitaries had ruptured capsule (eight out of 20 F344 and seven
out of 20 F344.BN-Edpm5BN ). Estrogen-treated F344.BNEdpm5BN rats have comparatively smaller tumors than F344
and these tumors have asymmetrical enlargement of pituitary
lobes with preserved pituitary tissue at places. Large areas of
necrosis are prominent in estrogen-treated F344.BN-Edpm5BN
pituitary and capsular break is seen. Hemorrhage is seen in all
cases, though it tended to be less severe in F344.BN-Edpm5BN
than in F344. One animal out of a sample of 20 estrogentreated F344.BN-Edpm5BN rats had a central pituitary infarct.
Neoplasia of the pituitary tumors of F344.BN-Edpm5BN rats
The neoplastic lesions in the pituitary (adenoma, adenocarcinoma) have been described as diffuse monomorphic cell populations with loss of lobular or adenomatous areas (8). Reticulin
staining (Figure 3) reveals that in both estrogen-treated F344
and estrogen-treated F344.BN-Edpm5BN rats, a true adenoma
is formed and the reticulin network is completely lost in
tumorous areas. Sheets of monomorphic cells are seen with
broken fragments of the reticulin fibers. Normal cellular
arrangement and architecture is lost with loss of normal reticulin pattern (Figure 3).
The cellular changes seen in both of the tumor-forming
strains (F344 and F344.BN-Edpm5BN ) are detailed in Table I.
Both of the tumor-forming strains show atypical neoplastic
cells in addition to the monomorphic cells. These are characterized by nuclear and cellular anaplasia. There are large and
hyperchromatic nuclei, nucleoli are prominent suggesting cell
replication and mitotic figures are seen in concordance with
earlier reports (3,8). Few abnormally large cells are seen. Most
of the tissue proliferation is seen in the outer zone with central
necrosis and fibrosis. Both F344.BN-Edpm5BN and F344
tumors, on microscopy have areas of cellular pleomorphism
and nuclear anaplasia, as well as invasion of capsule (Table I).
Effect of the BN allele of the Edpm5 QTL on microvasculature
and tumor-induced angiogenic activity
Although both F344 and BN rat pituitaries have an increase in
MVC upon chronic estrogen treatment, F344.BN-Edpm5BN rat
pituitaries do not (Figure 4). Therefore, inheritance of the BN
allele of the Edpm5 results in either a suppression of estrogeninduced angiogenesis, or a failure to induce angiogenesis in
response to estrogen. F344 rats show a statistically significant
(P 5 0.0005) increase in the MVC after treatment as reported
earlier (11). BN rats also show an increase in MVC after
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J.Pandey, A.Bannout and D.L.Wendell
Fig. 2. The F344.BN-Edpm5BN rat strain undergoes significant pituitary tumor growth upon chronic estrogen treatment and the tumors consist almost entirely of
lactotrophs. (A) Average pituitary mass of untreated and 10 weeks’ estrogen-treated rats of BN, F344 and F344.BN-Edpm5BN (abbreviated Edpm5) strains.
The estrogen-induced tumors of both (B) F344 and (C) F344.BN-Edpm5BN strains are prolactinomas as shown by anti-PRL immunohistochemistry. (D)
Lactotroph density, i.e. the total number of immunoreactive lactotrophs as a percentage of total pituicytes. Statistical significance of pairwise comparisons is
indicated by asterisk: P 5 0.05; P 5 0.005; P 5 0.0005. See Supplementary material online for a color version of this figure.
estrogen treatment, although the increase is of moderate statistical significance (P 5 0.05). This may correspond with
tissue remodeling ongoing with the increase in lactotroph
density of the BN strain (Figure 2). We have reported previously that analysis of vessel ultrastructure shows signs of
increased angiogenic activity and remodeling in the estrogentreated BN (11).
LM shows that after 10 weeks of chronic estrogen treatment,
pituitaries of F344.BN-Edpm5BN rats show areas of necrosis
along with infarction. In comparison, F344 pituitaries show
dilated and congested vessels in both control and treated rats.
In addition, hemorrhagic lakes are seen in estrogen-treated
F344 pituitaries as reported earlier (11). In the BN strain, the
pituitaries of neither untreated nor estrogen treated rats have
any significant abnormalities as seen by EM or by LM. This is
despite the lactotroph hyperplasia seen post-treatment, also
reported earlier (11). Pituitaries of untreated F344.BNEdpm5BN are similar to BN.
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Effect of BN allele of Edpm5 QTL on microvessel
ultrastructure and angiogenesis
Ultrastructure analysis of treated and control animals of all
three strains reveals prominent and distinct ultrastructural differences. Both parental strains (BN and F344) exhibit angiogenic activity after estrogen treatment with BN showing
moderate activity and F344 showing pronounced angiogenesis
(11). However, F344.BN-Edpm5BN pituitary vessels do not
show significant angiogenic activity after chronic estrogen
treatment.
Overall, pituitaries of untreated F344.BN-Edpm5BN and BN
do not show any significant angiogenic activity. However,
untreated F344 pituitaries do show features of ongoing angiogenesis. All genotypes have thin walled capillaries lined by
fenestrated endothelial cells (EC), with rounded or oval nuclei
and cells with uniform electron density. The capillary diameter
and perivascular spaces are variable. F344.BN-Edpm5BN and
Edpm5 prevents the angiogenic switch
Fig. 3. Reticulin pattern in the pituitary after 10 weeks of estrogen treatment. Untreated rat pituitary shows the normal reticulin pattern, where fiber network is
deposited around groups of cells forming a lobular or acinar pattern as seen in (A) untreated BN, (C) untreated F344 and (E) untreated F344.BN-Edpm5BN .
The pattern is similar and preserved in the case of (B) estrogen-treated BN pituitaries. In the case of estrogen-treated (D) F344 and (F) F344.BN-Edpm5BN
congenic rats however, a true adenoma is formed and the reticulin network is completely lost in tumorous areas. Sheets of monomorphic cells are seen with broken
fragments of the reticulin fibers. Necrosis and hemorrhage show up as brown amorphous areas. (Gomori’s Silver stain, magnification 600.)
BN pituitary vasculature is stable with negligible remodeling
activity seen. The EC has smooth abluminal boundaries with
discrete well-defined BM both on capillary and parenchymal
side. Capillaries have small perivascular spaces, which contain
thin wavy electron dense pericytes that are in close apposition
with the vessel walls and have processes intertwining with EC.
In contrast, F344 capillary ultrastructure shows features of
activation and ongoing vasculature remodeling or angiogenesis. There is instability of BM, with reduplication and loss at
places. Pericytes are plump and separated from the capillary
walls, lying in the opened up perivascular spaces. Evidence of
EC and pericyte migration is present with loss of vascular
integrity at places. Extravasation of RBCs into the tissue
spaces is seen, suggesting an increase in permeability and
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J.Pandey, A.Bannout and D.L.Wendell
Table I. Microscopic pituitary features of 10 weeks DES-treated rats
Rat strain
BN
Number of animals
Feature
Hyperplasia only
Adenoma:
with preserved areas
replacing the gland
Invasion of capsule
F344
F344.BN-Edpm5BN
6
6
6
Portion of animals within strain exhibiting feature
100%
0
0
0
0
0
50%
50%
50%
67%
33%
67%
Fig. 4. MVC after 10 weeks chronic estrogen treatment of rats of F344.BNEdpm5BN (abbreviated Edpm5) strain do not show any significant increase.
MVC are average/200 field. Statistical significance of pairwise
comparisons is indicated by asterisk: P 5 0.05; P 5 0.005;
P 5 0.0005.
leaky vessels. EC-pericyte interaction is patchy with loss of
BM between them. Intussusceptive angiogenesis is also seen in
F344 pituitaries.
Estrogen-treated rats of all three genotypes show variably
congested capillaries with RBCs, platelets and leukocytes. In
the case of estrogen-treated F344.BN-Edpm5BN rat pituitary
tumor however, most of the capillaries do not show features
pointing to significant vasculature remodeling or angiogenic
activity and are similar to those in the pituitaries of untreated
rats. The capillary network is less dense and typically has areas
of up to eight to 10 cell thickness without interlay of capillaries. In addition to this, areas of cellular necrosis are seen,
suggesting hypoxia due to lack of effective vasculature. At
most places pericytes and EC are in close apposition suggesting quiescent vasculature. Some vessels with reduplication of
BM and separation of pericytes are seen (Figure 5A), but these
are a rare finding. No migrating EC and/or pericytes are seen.
No significant degradation of extracellular matrix is seen outside of the necrotic areas. In contrast, pituitary tumors of
estrogen-treated F344 show pronounced angiogenic activity
as evidenced by organelle-rich EC, reduplication of BM is
present on both capillary and parenchymal side. BM is diffuse
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Fig. 5. (A) Ten weeks estrogen-treated F344.BN-Edpm5BN pituitary vessel
with reduplication of BM, separation of pericytes, increase in perivascular
space and EC projecting through the diffuse BM into pericapillary space
(arrow). This is a rare finding in the F344.BN-Edpm5BN strain, as there is
limited angiogenic activity. Magnification bar 5 mm. (B) Ten weeks
estrogen-treated F344 pituitary, shows opening of intercellular spaces due to
degradation of extra cellular matrix. A migrating endothelial cell with dense
lysosomes at the leading tip (arrow) and pericyte scaffolding is seen along
the sides (arrowhead). The parenchymal BM is largely intact, whereas the
capillary BM (upper right corner) shows disruption and reduplication.
Pericytes have detached from the capillary wall and can be seen migrating
into the opened up intercellular space (open arrow). Magnification
bar 2 mm.
at places and EC are seen protruding into the perivascular
spaces suggesting migration. Plump organelle-rich migrating
EC are seen in the pericapillary and intercellular spaces with a
pericyte scaffold (Figure 5B). At places they throw out processes that fuse to form lumens that contain RBCs suggesting
formation of new capillary lumens. Pericytes in all treated
F344 pituitaries are more numerous than controls and have
prominent nuclei and organelles and lie free in the perivascular
space. In addition to the above, estrogen-treated F344 show
microscopic lakes of RBCs bound by normal viable pituitary
epithelium with no vessel wall or BM. Estrogen-treated BN
pituitaries also show some angiogenic activity evidenced by
Edpm5 prevents the angiogenic switch
A
B
Fig. 6. The level of VEGF is significantly lower in pituitary of 10 weeks estrogen-treated F344.BN-Edpm5BN compared with estrogen-treated F344. (A) Levels of
VEGF, actin and PRL in pituitary homogenates of untreated and estrogen-treated rats of each strain. Each lane was a sample from a different rat. (B) Comparison
of the mass of the pituitary tumors of estrogen-treated F344.BN-Edpm5BN and F344 rats used for VEGF analysis. Bars correspond with the lanes of estrogentreated rats on the western blot above. (C) Quantitative comparison of average pituitary VEGF level, normalized to actin, in the pituitary of untreated and
estrogen-treated F344.BN-Edpm5BN and F344. Statistical significance of pairwise comparisons is indicated by asterisk: P 5 0.05; P 5 0.005.
EC sprouts and pericyte migration. BN also shows evidence of
intussusceptive angiogenesis (11). Thus, the F344.BNEdpm5BN strain has the lowest grade while F344 has the
most pronounced vasculature remodeling and angiogenic
activity.
Effect of BN allele of Edpm5 QTL on VEGF expression
It has been reported previously that the pituitary VEGF level is
increased by estrogen treatment in F344 rats (10), but not BN
(31). In the pituitary of untreated rats of both BN and F344,
VEGF level was not found to be different and was barely
detectable (31). In a previous QTL mapping study, we found
that a significant effect on pituitary VEGF level mapped to the
same portion of rat Chr5 where the Edpm5 QTL is located with
the BN alleles of these markers being associated with lowered
VEGF level in pituitary of estrogen-treated rats (31). The
nature of the data collected in this earlier study necessitated
the use of non-parametric statistics, which did not allow quantitative determination of the effect of the BN allele of Edpm5
on VEGF level. Now, with our congenic line, we can address
this more precisely. We find that VEGF levels in estrogentreated F344.BN-Edpm5BN rats are significantly lower than
F344 after chronic estrogen treatment (Figure 6). Estrogentreated F344.BN-Edpm5BN rats do have a small increase in
VEGF compared with untreated, but this difference is dwarfed
by the difference between estrogen-treated and -untreated
F344 (Figure 6). Thus, although there are several Edpm QTL,
all on different chromosomes, the presence of BN alleles of the
segment of rat Chromosome 5 that contains Edpm5 reduces
490% of the estrogen-induced increase in VEGF expression
that occurs in the F344 rat pituitary. VEGF level in the pituitary of untreated F344.BN-Edpm5BN rats is undistinguishable
from untreated F344 (Figure 6).
As described above, the pituitary tumor of estrogen-treated
F344.BN-Edpm5BN rats is quantitatively smaller than that of
F344. Therefore, to rule out the possibility that the difference
in VEGF expression was merely a consequence of reduced
tumor growth, we analyzed VEGF level in samples of rats for
which there was great overlap in the range of pituitary mass
(Figure 6B). In fact, the difference in average pituitary mass
between the samples of BN and F344 tumors selected for
VEGF analysis is not statistically significant. Despite this,
there is a dramatic difference in the level of VEGF.
Discussion
The tumor susceptible F344 strain has an inherent instability of
vasculature and in the presence of estrogen-induced hyperplasia readily responds by angiogenesis (2,9,11). In contrast, the
1835
J.Pandey, A.Bannout and D.L.Wendell
estrogen-treated F344.BN-Edpm5BN rats show negligible
angiogenic activity and vasculature remodeling despite incessant proliferation of lactotrophs and neoplasia. This means that
introgression of the BN allele of the Edpm5 QTL into F344 not
only corrects this inherent instability of vasculature, but also
prevents development of the angiogenic phenotype while
retaining the tumorigenic phenotype. Therefore, at least one
of the genes regulating a ‘switch’ to angiogenic phenotype
must reside at the Edpm5 QTL and the BN allele of this
gene(s) confers ability to suppress angiogenesis in the presence
of unabated estrogen stimulus and neoplastic transformation.
The F344.BN-Edpm5BN rat shows a distinct phenotype from
estrogen-treated rats of its parental strains, i.e. F344 (tumor
susceptible rat strain) and BN (tumor-resistant strain).
F344.BN-Edpm5BN rat pituitary tumor shows no increase in
MVC [a histological measure of angiogenesis (42,43)] after
estrogen treatment despite a significant increase in mass and
eventual tissue necrosis. It is known that tumors tend to compress their blood supply and this compression can lead to
cessation of blood flow in the core of the tumor leading to
necrosis (19). This appears to be true for estrogen-treated F344
rat pituitaries, where central necrosis is seen along with
increased microvessel count in the peripheral areas of pituitary
tumor. In the case of F344.BN-Edpm5BN rats, however, the
mechanism appears to be a true non-increase of vessels, as GS
lectin histochemistry would detect compressed or collapsed
blood vessels. Secondly the necrosis was not confined to the
core central part only, as seen in estrogen-treated F344 rats
(11). It was seen even in the peripheral adenomatous areas.
Angiogenic activity in response to estrogen treatment in the
F344.BN-Edpm5BN rat is even less than that observed in the
tumor-resistant strain as judged both by MVC and analysis of
ultrastructure.
The fact that the F344.BN-Edpm5BN strain’s angiogenic
phenotype, based on analysis of MVC and ultrastructure, differs from both the BN and F344 strains suggests that at least a
component of its effects could involve genetic interaction, i.e.
epistasis. The pituitary of estrogen-treated F344.BN-Edpm5BN
rats has even less angiogenic activity than that of estrogentreated BN, even though that strain is the source of the introgressed chromosome segment. Thus, the BN allele of Edpm5
appears to be having additional phenotypic effects in a different genetic background (F344). This is, by definition, genetic
interaction, though at present we do not have a mechanism for
how this interaction might occur. In contrast, for the quantitative trait of pituitary mass, the lower mass of the F344.BNEdpm5BN rat pituitary tumor compared with that of F344 is
consistent with the additive quantitative effect detected previously for Edpm5 by QTL mapping in an intercross design
(28). In that QTL mapping study, which initially identified
Edpm5, interaction tests were performed for the trait of pituitary mass but no statistically significant interactions were
detected between Edpm5 and markers for other QTL (28). A
way to reconcile these findings is to view the BN allele of
Edpm5 as having a main effect that is additive with an additional epistatic component.
Chronic estrogen treatment acting as a continuous mitogenic
stimulus ensures incessant lactotroph hyperplasia (2,3,38) and
resultant tumor formation, which is invasive (12), in the tumor
susceptible F344 strain (2,3). Estrogen may induce vascular
proliferation required for this process either by a direct effect
or indirectly. The estrogen-induced rat prolactinoma pathogenesis proceeds in two phases. In the first phase of chemical
1836
induction, estrogen acts directly on lactotrophs to stimulate
growth and then in the second phase of mechanical disinhibition leads to progression (46). This phase of progression must
require and promote angiogenesis seen in pituitary tumors
(17,47,48), as it is well established that progression from
hyperplasia to neoplasia requires the angiogenic switch in the
phenotype (21,49--51). The anterior pituitary gland contains a
dense network of capillaries, almost entirely derived from the
pituitary--hypothalamic portal system and does not contain any
arteries (52). Estrogen treatment of rats, however, has been
shown to induce arteriogenesis derived from overlying dural
vessels in tumor susceptible rats and to a small extent also in
tumor-resistant rats (9). This leads to a mechanical disinhibition (9) and along with the resultant hemodynamic changes
results in angiogenic activity (53,54) that leads to progression
of tumor. Estrogen thus influences the anterior pituitary vasculature causing vascular reorganization or angiogenesis. This
is highly pronounced in F344 (tumor susceptible) rat strain and
low grade in BN (tumor resistant) rat strain (2,11).
Light and electron microscopy studies have shown that during angiogenesis following a stimulus for neovascularization
(which in this case is estrogen-induced incessant proliferation
of lactotrophs), vascular permeability increases and ECs
change their morphology (55,56) and begin to degrade their
surrounding BM (56,57). Loss of vessel wall integrity with
fragmentation and reduplication of BM is seen. This facilitates
movement of cells from the intravascular compartment to
perivascular spaces. Pericytes are important in the initiation
and termination of angiogenesis (58,59) and their migration to
extravascular location is seen at the onset of new vessels
formation (56,60). Pericytes from perivascular locations
appear to guide newly formed sprouts through tissue (59) and
finally lead to their maturation and quiescence (58). Pituitaries
of estrogen-treated F344 rats show these changes consistent
with vessel remodeling and sustained angiogenesis as well as
atypical vessels in response to estrogen, as described earlier
(2,11). Extravasated RBCs are seen suggesting that vessel
integrity loss must be occurring early on (2,3,11). In treated
BN rats the changes are not very pronounced (11). In the case
of estrogen-treated F344.BN-Edpm5BN pituitaries, however,
the ultrastructure shows negligible vasculature remodeling
activity despite increase in mass and tumor formation, finally
leading to tissue hypoxia and necrosis. This suggests that
the F344.BN-Edpm5BN rat strain while unable to restrain the
estrogen-induced proliferative response, and thereby the adenoma formation, is able to either block or down regulate
angiogenesis possibly by preventing the ‘angiogenic switch’.
Hence the angiogenic response is inadequate and may lead to
the degenerative hypoxic effects seen in the form of cellular
necrosis. Thus, in F344.BN-Edpm5BN rats the process of neoplasia and the resultant and expected angiogenic response
(seen in F344 rats) have been uncoupled.
The expression of VEGF in the pituitary of estrogen treated
F344.BN-Edpm5BN rats demonstrates that the Edpm5 region
contains a major regulator of VEGF expression, though at this
point we cannot determine if its effect is direct or indirect.
However, the effects of Edpm5 must not be due to the mutation
of the VEGF gene itself because in the rat genome the Vegf
gene is present on chromosome 9 (61). Therefore Vegf can be
ruled out as a candidate gene for Edpm5. This finding also
suggests a mechanism for the lack of angiogenic activity in the
estrogen-induced tumor of the F344.BN-Edpm5BN strain.
However, it should be noted that VEGF also promotes vascular
Edpm5 prevents the angiogenic switch
permeability (32), which could be relevant to the hemorrhagic
lakes observed in F344 pituitary tumors (2,3,11), but absent in
F344.BN-Edpm5BN pituitary tumors.
A search of the rat genome of the portion of rat Chr5 that
contains Edpm5 and syntenic regions in the mouse, and human
genomes did not locate any compelling candidate genes, indicating that the phenotype of Edpm5 is either due to a novel
gene or a gene not implicated in angiogenesis previously. The
segment of rat Chr5 introgressed from BN into F344 to form
our congenic line extends from near the centromere-proximal
end to beyond the middle of the chromosome. Through a
search in February 2004 of the Rat Genome Database
(www.rgd.mcw.edu) for genes known to be on rat Chr5, and
of the Mouse Genome Informatics database (www.informatics.jax.org), and Human Genome (www.ncbi.org) in regions
that show synteny with rat Chr5, mouse Chr4 and human Chr9,
we found several genes associated with angiogenesis. These
were Id3 (62), Ece1 (63), Tek (a.k.a. Tie2) (64), and Wasf2
(a.k.a. WAVE2) (65). Id3 and Ece1 are both on the distal end of
rat Chr5 beyond the segment introgressed in our congenic line.
Tek and Wasf2 are all centromere proximal to the microsatellite marker D5Mit7 and thus within the chromosomal region
introgressed in our congenic line. However, they are all distal
to the previously mapped support interval of Edpm5 (28,31).
Although the switch to an angiogenic phenotype is important
to many tumor types, human pituitary adenomas are characterized by a paucity of angiogenesis and in fact have lower
microvessel counts than normal pituitary tissue (26). In this
regard, the pituitary tumor of the estrogen-treated F344.BNEdpm5BN rat with its characteristic of neoplasia without
increased angiogenic activity may be a more relevant model
for human prolactinoma than that of F344.
Supplementary material
Supplementary material can be found at: http://www.carcin.
oupjournals.org/.
Acknowledgements
Anti-rat PRL antibodies were provided by Dr A.F.Parlow of the NIDDK
National Hormone and Pituitary program. We are grateful to Dr Fay Hansen
for a range of research suggestions and providing GS Lectin and some other
reagents used. We also thank Parker Kelly, Maria Boardman, Tasnim Hoda for
a broad range of technical assistance. Todd Miller assisted in electron microscopy. Financial support was provided by Oakland University Research Excellence Program in Biotechnology and by National Institutes of Health, grant
# R01-CA 71911.
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Received December 1, 2003; revised April 20, 2004; accepted May 16, 2004