Mechanism of Dexamethasone Suppression of Brain Tumor–associated

Vascular Permeability in Rats

Involvement of the Glucocorticoid Receptor and Vascular Permeability Factor
John D. Heiss,* Efstathios Papavassiliou,* Marsha J. Merrill,* Lynnette Nieman,‡ John J. Knightly,* Stuart Walbridge,*
Nancy A. Edwards,* and Edward H. Oldfield*
*Surgical Neurology Branch, National Institute of Neurological Disorders and Stroke, and ‡Developmental Endocrinology Branch,
National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892

Abstract tumor cells act upon the tumor-associated vasculature. As a re-

sult, plasma components leak from the blood stream into the
Brain tumor–associated cerebral edema arises because tu- extracellular space of the brain, creating brain tumor–associ-
mor capillaries lack normal blood-brain barrier function; ated cerebral edema (2–4). This cerebral edema compounds the
vascular permeability factor (VPF, also known as vascular neurologic dysfunction associated with the underlying tumor.
endothelial growth factor, VEGF) is a likely mediator of Glucocorticoids reduce brain tumor–associated vascular
this phenomenon. Clinically, dexamethasone reduces brain permeability and cerebral edema, reduce intracranial pressure,
tumor-associated vascular permeability through poorly un- improve accompanying symptoms, and provide palliation of
derstood mechanisms. Our goals were to determine if sup- patients with malignant gliomas by prolonging useful neuro-
pression of permeability by dexamethasone might involve logic function and life span (5–7). Although dexamethasone is
inhibition of VPF action or expression, and if dexametha- a mainstay in the treatment of brain tumor patients, the mech-
sone effects in this setting are mediated by the glucocorti- anisms by which glucocorticoids reduce brain tumor–associ-
coid receptor (GR). In two rat models of permeability (pe- ated cerebral edema are poorly understood, and considerable
ripheral vascular permeability induced by intradermal morbidity is associated with their use. Iatrogenic Cushing’s
injection of 9L glioma cell–conditioned medium or purified syndrome, the result of high dose glucocorticoid therapy, is
VPF, and intracerebral vascular permeability induced by characterized by truncal obesity, glucose intolerance, myopa-
implanted 9L glioma), dexamethasone suppressed perme- thy, immunosuppression, hypertension, and adrenal suppres-
ability in a dose-dependent manner. Since 80% of the per- sion.
meability-inducing activity in 9L-conditioned medium was Whereas most of the side effects of glucocorticoid treat-
removed by anti–VPF antibodies, we examined dexametha- ment are presumed to be mediated by the glucocorticoid re-
sone effects of VPF expression in 9L cells. Dexamethasone ceptor (GR)1 (8, 9), it is not known whether the dexametha-
inhibited FCS- and PDGF-dependent induction of VPF ex- sone effect on brain tumor–associated edema is dependent on
pression. At all levels (intradermal, intracranial, and cell interaction with the GR. Doses of dexamethasone that are
culture), dexamethasone effects were reversed by the GR used clinically for treating brain tumor–associated cerebral
antagonist mifepristone (RU486). Dexamethasone may de- edema vary widely and far exceed those that saturate the pitu-
crease brain tumor–associated vascular permeability by two itary glucocorticoid receptors and suppress the hypothalamic–
GR-dependent mechanisms: reduction of the response of pituitary–adrenal axis. It has often been suggested that these
the vasculature to tumor-derived permeability factors (in- high doses are required because inhibition of cerebral edema
cluding VPF), and reduction of VPF expression by tumor occurs through a pharmacologic mechanism that does not re-
cells. (J. Clin. Invest. 1996. 98:1400–1408.) Key words: cere- quire the GR and that steroids lacking glucocorticoid effects
bral edema • vascular endothelial growth factor • glioma • might be appropriate in this capacity (10–16).
mifepristone • steroids Similarly, it is not fully understood at what level this dexa-
methasone effect on brain tumor–associated edema occurs: (a)
inhibition of the effects of permeability factors on the capillary
Introduction bed, and/or (b) inhibition of the production of permeability
factors by tumor cells. Vascular permeability factor (VPF, also
Brain tumor–associated vascular permeability arises because known as vascular endothelial growth factor, VEGF) has been
tumor capillaries lack the usual blood-brain barrier function of shown to be a likely mediator of increased vascular permeabil-
normal brain capillaries (1). This phenomenon is thought to ity in some models of peripheral tumors (17, 18), and the VPF
occur at least in part because permeability factors produced by secreted by glioma cells has been proposed to play the same
role in brain tumors (19, 20). Although VPF expression is
clearly increased in response to hypoxia in rat C6 glioma–
Address correspondence to Marsha J. Merrill, Surgical Neurology derived cells (21, 22), the regulation of VPF in glioma cells by
Branch, National Institutes of Health, 10 Center Drive, 10-5D37,
MSC 1414, Bethesda, MD 20892-1414. Phone: 301-496-6720; FAX:
301-402-0380.
Received for publication 2 August 1995 and accepted in revised 1. Abbreviations used in this paper: 9LCM, 9L cell conditioned me-
form 18 July 1996. dium; 14C-AIB, a [1-14C] aminoisobutyric acid; GR, glucocorticoid re-
ceptor; K, blood-to-brain transfer constant; KLH, keyhole limpet
The Journal of Clinical Investigation hemocyanin; recVPF, recombinant human VPF; RU486, mifepris-
Volume 98, Number 6, September 1996, 1400–1408 tone; VPF, vascular permeability factor.

1400 Heiss et al.

other inducers or by dexamethasone has not been well studied, 9L cell conditioned medium. 9L gliosarcoma cells were grown in
although evidence exists that such regulation may occur in hu- DME, 10% FCS, 50 U/ml penicillin, and 50 mg/ml streptomycin to
man glioma cells (19, 23). In addition, dexamethasone has near confluence, washed twice with PBS, and changed to serum-free
been shown to interfere with the early effects of VPF on en- DME. After 6 d, medium was exhaustively dialyzed against a solution
of 0.25 M ammonium bicarbonate (adjusted to pH 7.4 using glacial
dothelial cells (24).
acetic acid) using a Spectra/Por 6, 25-kD membrane (Spectrum Medi-
These observations emphasize the need for experimental cal Industries, Inc., Laguna Hills, CA). The dialyzed solution was fro-
studies that define the mechanism of action and the optimal zen, lyophilized, and redissolved in PBS. Pilot studies demonstrated
dosing regimens for glucocorticoid treatment of brain tumor– that all of the permeability-inducing activity in 9LCM was retained by
associated cerebral edema. Further, knowledge of the relative this procedure. The protein concentration in PBS solutions contain-
importance of GR- vs non–GR-mediated effects is essential to ing lyophilized conditioned medium was determined by the Bio-Rad
predict if compounds such as the 21-aminosteroids, which have Protein Assay (Bio-Rad Laboratories, Richmond, CA).
a structure similar to dexamethasone but lack receptor-medi- Miles assay. The Miles assay uses intradermal injection of test
ated side effects, will be effective in reducing brain tumor– substances and intravascular injection of Evans blue dye (which binds
associated cerebral edema (15, 25). Such knowledge should to endogenous serum albumin) as a tracer to assay permeability in
peripheral vessels. The assay was performed essentially as described
provide rational guidelines for optimizing the use of steroids in
(37), and all animal procedures were performed in accordance with
the treatment of brain tumor–associated cerebral edema while the National Institutes of Health (NIH) Animal Care and Use Guide-
limiting side effects, and be helpful for the development of lines. Fisher 344 rats (250–300g; Taconic Farms Inc., Germantown,
novel therapeutic approaches to this disease. NY) were given intraperitoneal injections of steroid or carrier. 6 h
These studies were undertaken to elucidate the mecha- later, the animals were anesthetized using 90 mg/kg intraperitoneal
nisms by which dexamethasone suppresses brain tumor–asso- ketamine and 10 mg/kg Xylazine, and, after removing a blood sample
ciated cerebral edema, particularly with respect to the roles of for corticosterone level, given intracardiac injection of 30 mg/kg 2%
the GR and VPF. To address these issues, we used the 9L Evans blue (Sigma Chemical Co.) in PBS. Intradermal 0.1-ml injec-
gliosarcoma brain tumor model in the rat. This model was cho- tions of 9LCM, recombinant human VPF (rec VPF), or PBS (back-
sen because of several relevant features. 9L cells secrete VPF ground) were placed on the dorsal skin in rows of three. The animals
were killed 30 min after injections and the skin removed. Wheals
both in culture and in vivo (22, 26). The ability of the 9L tumors
were resected using an 8-mm diameter trephine (Roboz Surgical In-
implanted in the rat brain to increase brain tumor–associated strument Co., Inc., Rockville, MD) and incubated in 2 ml formamide
vascular permeability and brain edema is well documented (Fluka AG, Buchs, Switzerland) at 458C for 48 h. OD of the extracts
(27–31). Dexamethasone reduces brain tumor–associated ce- was read at 620 nM in a spectrophotometer (6/20; Coleman, Oak
rebral edema in the 9L model as well as in humans (31–36). Brook, IL).
We examined the effects of dexamethasone on three parame- Anti–VPF antibodies. A peptide corresponding to the 25 NH2-
ters: (a) the ability of 9L cell conditioned medium (9LCM) or terminal amino acids of mature rat VPF (26) was synthesized and
purified VPF to induce vascular permeability in peripheral coupled to the carrier protein keyhole limpet hemocyanin (KLH) by
vessels (Miles assay); (b) the expression of VPF mRNA by 9L 0.2% glutaraldehyde (38). Antibodies were raised in rabbits both to
cells in culture; and (c) the degree of vascular permeability as- the VPF-KLH conjugate (anti–VPF) or to KLH alone (control anti-
serum). Adsorption of VPF from 9LCM was performed by incubating
sociated with 9L tumors implanted in the brain using a [1-14C]
9LCM with antibodies bound to protein A agarose (Pierce Chemical
aminoisobutyric acid (14C-AIB) as a tracer. To determine to Co., Rockford, IL) as described previously (20).
what extent the effects of dexamethasone involve the GR, we VPF expression in 9L cells. 9L cells were plated in T–75-cm2
examined the reversibility of the dexamethasone effects by the flasks and grown to z 75% confluency, washed twice in PBS, and
GR antagonist mifepristone (RU486). We report that suppres- changed to serum-free DME. After 48 h, the culture medium was
sion of brain tumor–associated cerebral edema by dexametha- changed to fresh medium containing the additions indicated in the
sone may occur through actions both on the tumor cell and on figure legends. For cells exposed to hypoxia, medium was first de-
the associated vasculature, and that these effects are GR-medi- gassed under vacuum for several hours. A 5% CO2/95% N2 mixture
ated and involve VPF. was bubbled through this medium and the medium transferred to
flasks that were also flushed with the gas mixture and immediately
sealed. Analysis of the medium confirmed hypoxic conditions (PO2 of
Methods z 20 vs z 150 mmHg for normoxia). After 6 h, cells were harvested
and RNA isolated according to Chomczynski and Sacchi (39). RNA
Reagents. Dexamethasone sodium phosphate was obtained from The was subjected to electrophoresis and transfer as previously described
Butler Co. (Columbus, OH) for use in animals, and from Sigma Chem- (40). Blots were probed using the insert from a Bluescript vector con-
ical Co. (St. Louis, MO) for use in cell culture. RU486 (17b-hydroxy- taining rat VPF164 cDNA (41) or a mouse actin probe (Ambion Inc.,
11b-[4-dimethylamino phenyl]-17a-[1-propynyl]estra-4,9-dien-3-1) was Austin, TX). Probes were labeled with 32P in a random primer exten-
graciously provided by Roussel, UCLAF (Paris, France). The 21-ami- sion reaction and hybridized using Quik Hyb (Stratagene Inc., La
nosteroids, tirilazad mesylate (U-74006F, 21-[4-(2,6-di-1-pyrrolidinyl- Jolla, CA) according to the manufacturer’s instructions. Consistent
4-pyrimidinyl)-1-piperazinyl]-16a-methyl-pregna-1,4,9(11)-triene-3,20 results were obtained from two or three separate experiments.
dione monomethanesulfonate), and U-74500A (21-{4-[5,6-bis (diethyl- Brain tumor model. The 9L brain tumor model was established
amino)-2-pyridinyl]-1-piperazinyl}-16a-methyl-pregna-1,4,9(11)-triene- as previously described (42). Briefly, Fisher 344 rats were anesthe-
3,20 dione hydrochloride) were graciously provided by The Upjohn Co. tized using intraperitoneal ketamine and Xylazine and placed in a ste-
(Kalamazoo, MI). DME, penicillin–streptomycin, and fetal calf serum reotaxic apparatus (David Kopf Instruments, Tujunga, CA). Synge-
were supplied by Gibco Laboratories (Grand Island, NY). Recombinant neic 9L gliosarcoma cells (5 3 104) in 5 ml PBS were injected into the
human VPF and PDGF-BB were purchased from R & D Systems, Inc. white matter (coordinates, 1 mm anterior and 3 mm lateral to the
(Minneapolis, MN). Ketamine was obtained from Fort Dodge Labo- bregma; depth of inoculation, 3.5 mm) of the right cerebral hemi-
ratories (Fort Dodge, IA). Xylazine was supplied by the Mobay Corp. sphere using a 10-ml Hamilton syringe connected to the manipulating
(Shawnee, KS). Serum corticosterone levels were measured by radio- arm of the stereotaxic apparatus. Animals were randomly divided
immunoassay (Hazleton Laboratories, Vienna, VA). into treatment groups on day 16, at which time animals were given in-

Dexamethasone Suppression of Brain Tumor-associated Permeability 1401

traperitoneal injections of steroids or control solution every 8 h for 48 h. inability of this antibody to inhibit all of the permeability activ-
On day 18, the right femoral artery and vein were cannulated with the ity is probably due either to the presence of other permeability
animal under anesthesia. After obtaining a baseline blood sample, factors in the 9LCM or to the inability of the antibody to rec-
100 mCi of 14C-AIB (American Radiolabeled Chemicals, St. Louis, ognize all of the potential VPF conformations.
MO) was injected into the femoral vein. Arterial blood samples were
Dexamethasone suppression of permeability induced by 9L
obtained at 0.25, 0.5, 0.75, 1, 2, 3, 4, 5, 10, and 15 min and centrifuged
to obtain serum for scintillation counting. After the last blood sam-
conditioned medium and VPF in peripheral vessels. Initial ex-
ple, animals were killed, and the brain was removed, covered with an periments were designed to determine the concentrations of
embedding matrix containing polyvinyl alcohol and polyvinyl glycol 9LCM required for induction of vascular permeability in the
(O.C.T. compound; Miles Laboratories Inc., Elkhart, IN), and rapidly
frozen in a beaker of 2-methylbutane (EM Science, Gibbstown, NJ)
in dry ice (43).
Quantitative autoradiography. Frozen tissue sections were pre-
pared on the cryotome (Microm, Heidelberg, Germany). 10-mm his-
tologic sections were stained with hematoxylin and eosin. 20-mm au-
toradiographic sections were placed on Bainbridge Board (Charles T.
Bainbridge’s Sons, Cranbury, NJ) along with radioactive standards
(14C-plastic autoradiographic standards calibrated by the Laboratory
of Cerebral Metabolism, National Institute of Mental Health [Be-
thesda, MD] to 20-mm brain sections containing known amounts of
14
C) (44). The sections were placed in a cassette with SB-5 film (East-
man-Kodak Co., Rochester, NY) for 6 d and were evaluated using the
method of Hiesiger et al. (43). The outlines of cortex and tumor, as
determined from inspection of the histologic sections, were superim-
posed on the corresponding autoradiographic images to define the
boundary of the tumor on the autoradiogram. After calibrating the
equipment to a reference standard of 1,000 pixels per mm2, the area
within the tumor outline was measured and the mean tumor diameter
calculated. The 14C-AIB radiographic standards were used to convert
the optical density of the autoradiographic images to nCi/g. The ra-
dioactivity, nCi/g, of the tumor was measured and the unidirectional
blood-to-brain transfer constant (K) for the tumor was calculated by
dividing the tumor concentration of 14C-AIB by the integral of the
plasma concentration of 14C-AIB from 0 to 15 min (43).

Results
Neutralization of 9L glioma permeability activity by anti–VPF
antibodies. 9L cells produce VPF in culture and when im-
planted in vivo (22, 26). However, the extent to which VPF is
responsible for the permeability activity produced by 9L cells
in not known. To address this question, we incubated 9LCM
with anti–VPF antibodies and assayed for permeability activity
using the Miles assay. Although the Miles assay is not com-
pletely representative of the brain tumor situation, it is a useful
method for analyzing the nature, effects, and regulation of tu-
mor-derived permeability factors. Approximately 80% of per-
meability activity was removed by this antibody (Table I). The

Table I. Adsorption of Permeability Activity from 9LCM by

Anti–VPF Antibodies
Figure 1. Effect of dexamethasone on induction of cutaneous vascu-
Antiserum Permeability Percent, relative to control lar permeability and on corticosterone levels. (A) Animals were given
0.1-ml intradermal injections of 9LCM ( ) or recVPF ( ). 1 U
None 26.566.8 100 protein injected equaled 5 mg for 9LCM and 1 ng for recVPF. Perme-
Anti–VPF 5.661.4 21* ability was determined by the Miles assay and expressed as OD620 3
Anti–KLH 28.268.8 106‡ 100. Each point represents mean6SEM of nine 9LCM or four
recVPF animals. (B) Rats were given a single intraperitoneal injec-
tion of dexamethasone (controls received PBS) at the indicated dose.
9LCM was incubated with immobilized IgG from either control (anti– 6 h later, animals were given intradermal injections of 45 mg 9LCM,
KLH carrier) or anti–VPF (VPF peptide1KLH) antiserum. Control 10 ng recVPF, or PBS as indicated. Permeability was determined by
9LCM or 9LCM incubated with the indicated antibody was assayed for the Miles assay. Each point represents mean6SEM of 4–10 animals.
activity in the Miles assay. Permeability is expressed as OD620 3 100. (C) Rats were given a single dose of dexamethasone as above. 6 h
Data are expressed as the mean6SD (n 5 3). Background values (PBS) later, blood for corticosterone determination was obtained by cardiac
have been subtracted. *Significantly different from control (P 5 0.006); puncture immediately before performance of the Miles assay. Bars
‡
not significantly different from control (P 5 0.8). represent the mean6SD of seven to eight animals.

1402 Heiss et al.

 Figure 2. Effect of tein, up to 10 ng of recVPF (Fig. 1 A). Further experiments
RU486 on dexametha- were done within the linear range to allow easy detection of
sone inhibition of cuta- suppression of permeability by steroids. To establish whether
neous permeability. dexamethasone inhibits the permeability induced by intrader-
Rats received a single
mal injection of 9LCM or recVPF, rats were treated with vari-
intraperitoneal injec-
ous doses of dexamethasone 6 h before the Miles assay. Previ-
tion of control (PBS),
dexamethasone (Dex), ous work in our laboratory demonstrated effective suppression
RU486 (RU), or Dex1 of permeability with dexamethasone given 6 h before the
RU as indicated. The Miles assay in guinea pigs (19). The vascular permeability in-
dexamethasone dose duced by either 9LCM or recVPF was reduced in a dose-depen-
was 0.075 mg/kg in all dent manner by dexamethasone (Fig. 1 B). A dexamethasone
cases. (A) RU486 was dose of 0.1 mg/kg produced z 70% reduction in the perme-
given at 13, 103, or ability induced by 9LCM. A dexamethasone dose of 0.075 mg/
1003 dose (0.075, 0.75, kg was established as the ED50 for suppression of 9LCM-induced
or 7.5 mg/kg, respec-
permeability. Vascular permeability induced by recVPF was at
tively) as indicated. 6 h
least as easily suppressed as that induced by 9LCM and is con-
later, cutaneous perme-
ability was induced by sistent with the hypothesis that VPF is the predominant per-
intradermal injection of meability-enhancing agent in 9LCM. Dexamethasone suppres-
9LCM (18 mg/injection) sion of the permeability response to conditioned medium is
and permeability was also quantitatively similar to suppression of permeability in-
determined by the duced by histamine (data not shown), indicating that dexa-
Miles assay and ex- methasone suppression of permeability is not necessarily spe-
pressed as OD620 3 100. cific to the permeability-enhancing agent.
Background values (intradermal injection of PBS) have been sub- Suppression of serum corticosterone by dexamethasone. If
tracted. Bars represent mean6SEM of five to nine animals. *Signifi-
dexamethasone suppression of vascular permeability is medi-
cantly different from Dex alone (P 5 0.02), not significantly different
ated by the GR, then the dose-response curve should mimic
from controls; **not significantly different from control or Dex 1
1003RU. (B) As above except that permeability was induced by in- the curve for a known GR-mediated effect of dexamethasone,
jection of 5 ng recVPF. Bars represent mean6SEM of four animals. namely suppression of serum corticosterone levels. Therefore,
*Significantly different from Dex alone (P 5 0.015), not significantly we examined dexamethasone suppression of serum cortico-
different from controls; **not significantly different from control or sterone levels in these rats (Fig. 1 C). The dose-response curve
Dex11003RU. for suppression of Evans blue extravasation in the Miles assay
correlated with suppression of corticosterone levels (r 5 0.77,
P , 0.001), suggesting that both processes are receptor medi-
Miles assay. Since z 20% of the permeability activity in 9LCM ated.
was not removed by the anti–VPF antibody, recombinant VPF Reversal of dexamethasone effect by RU486. With the use of
alone was also included for comparison with the 9LCM. The RU486, a GR antagonist, it is possible to block the GR-mediated
dose-response curve was linear at least to 45 mg of 9LCM pro- effects of dexamethasone in experimental animals. To examine

Figure 3. Effects of FCS, dexamethasone, and RU486 on VPF expression in 9L cells. 9L cells were grown to z 75% confluency and changed to
serum-free medium. After 48 h, additions were made simultaneously as indicated. FCS was added to a final concentration of 10%. Cells were
harvested after 6 h and total RNA isolated. Northern blots (20 mg RNA) were analyzed using a probe for rat VPF and mouse actin. (A) Cells
were cultured in the presence of increasing concentrations of dexamethasone in either the absence (Control) or presence of FCS. Dexametha-
sone concentration is indicated below. (2) Indicates the absence of dexamethasone. (B) FCS was added to all cells along with steroids as indi-
cated. In this experiment, where added, dexamethasone (DEX) was present at a final concentration of 1027 M. Increasing concentrations of
RU486 were added as indicated. (2) Indicates the absence of RU486.

Dexamethasone Suppression of Brain Tumor-associated Permeability 1403

 Figure 4. Effect of PDGF of VPF
expression in 9L cells. Cells were
treated as described in Fig. 3. Cells
were harvested 6 h after the indi-
cated additions. Northern blots (20
mg RNA) were analyzed using a
probe for rat VPF and mouse actin.
(A) Cells received no additions
(Ctrl), 10% FCS, or 1, 10, or 100 ng/
ml PDGF. (B) Cells received no in-
ducer (Ctrl), 10 ng/ml PDGF, or
10% FCS. Additions were made si-
multaneously with (1) or without
(2) 1027 M dexamethasone.

directly whether dexamethasone effects were GR mediated, we was of interest to determine directly the effect of PDGF on VPF
used RU486 to determine if dexamethasone suppression of expression in these cells. PDGF induced expression of VPF
9LCM-induced permeability depended on activation of the mRNA in 9L cells in a dose-dependent manner (Fig. 4 A). As
GR. Rats were treated with dexamethasone ED50 (0.075 mg/ with the FCS-dependent induction of VPF mRNA, VPF induc-
kg), either alone or with RU486 at doses 1, 10, or 100 3 tion by PDGF was also inhibited by dexamethasone (Fig. 4 B).
greater than dexamethasone. Dexamethasone ED50 was cho- Hypoxia has also been reported to increase VPF in 9L cells
sen to enhance sensitivity to the effects of GR antagonism by (22) and is thought to be relevant to induction of VPF in hu-
RU486. RU486 reversed the antipermeability effect of dexa- man tumors (21, 51). However, unlike the FCS-dependent in-
methasone when given in doses 100 3 greater than dexameth- duction of VPF, dexamethasone failed to inhibit the VPF in-
asone, but not in doses 1 or 10 3 greater (Fig. 2 A). RU486 alone duction occurring in 9L cells under hypoxic conditions (Fig. 5
did not affect permeability. The antagonism of the dexameth- A), suggesting that the increase in VPF by FCS and hypoxia
asone effects by RU486 at 1003 concentration is consistent with may be occurring through different mechanisms in these cells.
previous results demonstrating RU486 inhibition of known re- In addition, the level of VPF observed in the presence of both
ceptor-mediated effects (45, 46). The RU486 experiments were hypoxia and FCS is greater than the level observed in the pres-
also performed using recVPF as the permeability-inducing ence of either inducer alone (Fig. 5 B). Although dexametha-
agent. The dexamethasone suppression of recVPF activity was sone was unable to suppress the increase in VPF expression
also reversed by RU486 (Fig. 2 B), which is consistent with the that occurs in the presence of hypoxia alone, dexamethasone
hypothesis that dexamethasone inhibition of VPF-induced was able to partially suppress the induction that occurred in
permeability requires the GR. the presence of both hypoxia and FCS (Fig. 5 C), presumably
Effects of dexamethasone on VPF expression in 9L cells. by inhibiting the FCS-mediated component of the VPF in-
Dexamethasone has been shown in some normal cell types to crease as observed above.
inhibit the induction of VPF mRNA that occurs in response to Suppression of vascular permeability in the 9L brain tumor
tumor promoters (47, 48). Since VPF is responsible for at least by dexamethasone. If the observed effects of dexamethasone
80% of the permeability activity produced by 9L cells, a dexa- on peripheral vessel permeability and on 9L cells in culture are
methasone-dependent inhibition of VPF expression in glioma
cells provides another possible mechanism by which steroids
might reduce vascular permeability in brain tumors. Other
than hypoxia (22), the influence of other possible regulators of
VPF expression in 9L cells has not been examined. Therefore,
we examined VPF mRNA levels in the presence of FCS and
dexamethasone. VPF mRNA was markedly upregulated in re-
sponse to FCS (Fig. 3 A). In the presence of dexamethasone,
basal levels of VPF were not affected, but the ability of FCS to
induce VPF mRNA was inhibited in a dose-dependent manner
(Fig. 3 A). Inhibition was nearly complete by 1027 M dexameth-
asone. To determine whether or not this effect of dexameth-
asone also occurs through a GR-mediated mechanism, we at-
tempted to reverse the dexamethasone-dependent inhibition Figure 5. Effect of dexamethasone and FCS on induction of VPF by
of VPF induction with RU486. RU486 blocked the dexa- hypoxia. Cells were treated as described in Fig. 3. Northern Blots (20
methasone inhibition of FCS-mediated VPF induction (Fig. 3 mg RNA) were analyzed using a probe for rat VPF and mouse actin.
B), indicating that the dexamethasone-dependent inhibition of Hypoxic induction was carried out as described in Methods. Control
cells (Ctrl) were maintained in a normoxic environment and received
FCS-mediated VPF induction occurs via a mechanism requir-
no additions. (A) Cells were maintained in a hypoxic environment in the
ing the GR. RU486 alone did not affect the FCS-dependent in- absence (Hyp) or presence (Hyp,Dex) of dexamethasone (1027 M)
duction of VPF expression (Fig. 3 B) or the basal level of VPF for 6 h. (B) Cells were exposed to either hypoxia (Hyp) or FCS alone,
expression (data not shown). or to both inducers simultaneously (Hyp,FCS) for 6 h. (C) As in B ex-
Since PDGF is a major mitogen in serum and has been pro- cept that some cells received dexamethasone simultaneously with ex-
posed as an important growth factor in brain tumors (49, 50), it posure to both hypoxia and FCS (Hyp, FCS, Dex).

1404 Heiss et al.

relevant to brain tumors in vivo, then dexamethasone suppres- volved in the dexamethasone suppression of brain tumor–asso-
sion of vascular permeability in the tumor-associated vascula- ciated vascular permeability. To examine more directly the im-
ture should also be reversible by RU486. The 9L homograft portance of the receptor-mediated effects of dexamethasone
brain tumor model using the amino acid analogue 14C-AIB as a on brain tumor–associated vascular permeability, RU486 was
tracer quantifies vascular permeability in the tumor-bearing given with dexamethasone to tumor-bearing animals and per-
brain by determining the blood-to-brain transfer constant for meability was assessed, as described above. This experiment
AIB (52). Despite the widespread use of the 9L brain tumor was performed with animals implanted at the same time as
model for the study of brain tumor–associated permeability those used to establish the dose–response relationship with
and brain tumor–associated cerebral edema (27–30, 53) and its dexamethasone. Animals were treated with 100 mg/kg per d
inhibition by dexamethasone (31, 54), a dose–response curve RU486 or 1 mg/kg per d dexamethasone alone, or with those
for this dexamethasone effect had not been established previ- doses of RU486 and dexamethasone together (Fig. 6). RU486
ously. To establish this curve, tumor-bearing rats received the reversed the effect of dexamethasone on permeability (P 5
indicated dose of dexamethasone for 48 h before the measure- 0.008), but had no effect on permeability when given alone.
ment of capillary permeability. Treatment of rats with increas- Effect of 21-aminosteroids on brain tumor–associated vas-
ing doses of dexamethasone produced a dose-dependent de- cular permeability. The 21-aminosteroids are structurally simi-
crease in 14C-AIB blood-to-brain tumor transfer (Fig. 6). lar to glucocorticoids, but lack their GR effects. To assess the
Dexamethasone at 1 mg/kg per d produced a 47% reduction in effect of the 21-aminosteroids on brain tumor–associated per-
the transfer constant compared to control (P 5 0.003). Increas- meability, animals were treated with either U-74006F (Tir-
ing the dose to 10 mg/kg per d had an additional effect, pro- ilazad) or U-74500A instead of dexamethasone, and perme-
ducing a 60% reduction in K. Dexamethasone treatment did ability was assessed by the 14C-AIB method as above. To
not affect tumor size. Control tumor mean diameter was improve detection of any effect on permeability, 10 mg/kg per
5.461.6 mm and that of dexamethasone-treated animals was d 21-aminosteroid, equal by mass to the highest dose of dexa-
5.461.9 mm (0.1 mg/kg per d), P 5 0.9; 5.961.7 mm (1.0 mg/kg methasone used in our 9L brain tumor trials, was chosen. Nei-
per d), P 5 0.6; and 6.060.8 mm (10 mg/kg per d), P 5 0.5. The ther 21-aminosteroid suppressed permeability (Fig. 6), suggest-
histologic appearance of the 9L tumor, a solid core of tumor ing a requirement for the GR in dexamethasone suppression of
cells with finger-like projections reaching into the surrounding permeability. In fact, U-74500A treatment resulted in an in-
brain, was identical in the control and dexamethasone-treated creased transfer constant (K 5 5.8661.88) compared to con-
groups. trol (K 5 3.5960.92, P 5 0.01). Treatment with 21-aminosteroids
Effect of RU486 on dexamethasone effects in brain tumors. did not affect tumor size; control tumor mean diameter was
The higher doses of dexamethasone required to inhibit perme- 5.461.6 mm and that of U-74006F–treated animals was 6.46
ability in the brain tumor model compared with the peripheral 0.6 mm, P 5 0.2, and that of U-74500A–treated animals was
model suggests that non–GR-mediated mechanisms may be in- 5.560.4 mm, P 5 0.6.

Discussion
That dexamethasone reduces brain tumor–associated edema
in animal models and clinical settings is well known. However,
the mechanisms by which this occurs are poorly understood.
Our results suggest that dexamethasone suppression of vascular
permeability in the rat 9L brain tumor model may occur through
two mechanisms: inhibition of the effects of tumor-derived per-
meability factors on the vascular bed, and inhibition of the
production of VPF by tumor cells. Reversibility of these dexa-
methasone effects by RU486 demonstrates the requirement at
both levels for the GR.
Evidence is mounting that VPF secretion by glioma cells
may underlie the increased vascular permeability associated
with these tumors. VPF and its receptors are upregulated in
both rodent and human brain tumors (20–22, 55). The major
inducer of permeability contained in 9L conditioned medium
is VPF (this report). Previous work from our laboratory dem-
Figure 6. Effect of dexamethasone, RU486, and 21-aminosteroids on onstrated that 75-99% of the cutaneous permeability response
brain tumor vascular permeability. Rats were implanted with 9L tu- to human glioma cell–conditioned medium and to glioma cyst
mor cells as described. On day 16, intraperitoneal administration of fluids is also blocked by an antibody to VPF (20). Therefore,
dexamethasone (Dex) and/or RU486 (RU) was begun at the indi- elucidation of the mechanisms by which VPF is regulated in
cated dose (mg/kg/d) and was given in divided doses every 8 h for 2 d.
these tumors is an important task. The VPF gene contains an
The 21-aminosteroids, U-74006F and U-74500A, were given at 10 mg/
AP-1 binding site (56), and VPF increases in response to sub-
kg per d as described above. 14C-AIB infusions were done on day 18
and the transfer constant was determined as described in Methods. stances that interact with that site such as serum, growth fac-
Values represent mean6SD of four to nine animals. Values signifi- tors (including PDGF), and tumor promoters (47, 48, 57). VPF
cantly different from control: *P 5 0.003, **P 5 0.001, ****P 5 also increases in response to serum in some human glioma cell
0.012; ***value significantly different (P 5 0.008) from 1 mg/kg per d lines (23) and in rat 9L cells (this report). We demonstrate
dexamethasone alone. here that induction of VPF expression by this mechanism is in-

Dexamethasone Suppression of Brain Tumor-associated Permeability 1405

hibited by dexamethasone, and that the inhibition is reversed ability factors other than VPF, the effects of which are not sup-
by the GR antagonist RU486. Dexamethasone inhibition of VPF pressed by dexamethasone. Interestingly, in the Miles assay,
induction most likely results from binding of the dexameth- suppression at 0.5 mg/kg, dexamethasone was more complete
asone–GR complex to the AP-1 transactivator complex, which (100% vs 88%, P 5 0.10) for recVPF- than for 9LCM-induced
in turn prevents the AP-1 transactivator complex from stimu- permeability, raising the possibility that the small percentage
lating transcription of genes containing an AP-1 site (58, 59). of permeability-enhancing activity of 9LCM that is insensitive
Although this effect of dexamethasone does not require GR- to dexamethasone suppression may not be due to VPF. (d) At
dependent gene transcription, it does require the binding of a given dose of systemically administered dexamethasone, the
dexamethasone to the GR. brain level of dexamethasone is lower than that in the periphery.
VPF is also induced by hypoxia in these cells, but this in- Several findings support the explanation that the relative
duction was not inhibited by dexamethasone. This is in agree- insensitivity to dexamethasone of the brain tumor model, com-
ment with previous results from NIH 3T3 cells (48), and sug- pared to the Miles assay, may be a result of the relative imper-
gests that induction by hypoxia in these two cell lines may meability of the brain to dexamethasone (64, 65). Tamargo, et
result from an increase in VPF mRNA stability. This process is al. (31) demonstrated that suppression of cerebral edema in
independent of the AP-1 site and therefore not affected by the 9L tumor model depends more on brain dexamethasone
dexamethasone (48). A hypoxia-mediated increase in VPF levels than on plasma levels, since dexamethasone must pene-
mRNA stability also occurs in retinal epithelial cells (60). That trate the blood-brain barrier to suppress permeability in that
VPF induction by FCS and hypoxia are occurring through dif- model. They reported that even after 8 d of systemic adminis-
ferent mechanisms in 9L cells would explain why the two in- tration of dexamethasone, levels of the steroid in the cerebral
ducers increase VPF more than either one alone and why dexa- hemisphere containing a 9L tumor were only 4% of those in
methasone only partially inhibits the increase observed in the the plasma (31). Our finding that suppression of pituitary
presence of both inducers. However, in another glioma cell ACTH secretion and 9L-conditioned, medium-induced dermal
line, hypoxia had been shown to increase VPF mRNA through vascular permeability occurs with dexamethasone doses that
transcriptional activation (61). In addition, expression of Fos have little effect on the brain is consistent with the finding of
and Jun (components of the AP-1 transactivator complex) and Miller et al. (66) that dexamethasone activates rat pituitary
subsequently VPF are upregulated by hypoxia in a hepatoma GRs (type-II adrenal steroid receptors) at blood concentra-
cell line (62), in which case dexamethasone suppression of hy- tions that do not activate GRs in the brain, even though the in-
poxia-mediated VPF induction is certainly possible. Since the herent affinity of the brain and pituitary GR for dexametha-
regulation of VPF by hypoxia, growth factors, and dexametha- sone in vitro is the same. Our finding that the dexamethasone
sone has not yet been studied in human glioma cells, the extent effect in both models is reversible by RU486 also suggests that
to which dexamethasone decreases vascular permeability in the higher doses of dexamethasone required to activate the
brain tumor patients through an inhibition of VPF production brain GR, and subsequently reduce brain tumor–associated
remains to be determined. It is of interest that PDGF induces vascular permeability, is at least partially explained by inade-
VPF mRNA in 9L cells and that this induction is inhibited by quate dexamethasone entry into the brain at lower doses.
dexamethasone. Since PDGF expression is often elevated in The essential role of the GR in mediating the inhibition of
human gliomas (50, 63), it is possible that PDGF also induces vascular permeability by dexamethasone is further supported by
VPF expression in the human setting, and that this expression the finding that the 21-aminosteroids, compounds with a glu-
may be inhibited by dexamethasone administration. cocorticoid structure but without GR-mediated effects, were
We have demonstrated that the GR is the common media- ineffective in suppressing permeability. The 21-aminosteroids, or
tor of dexamethasone inhibition of FCS-dependent VPF in- lazaroids, possess the non–GR-mediated effects of methyl-
duction in 9L cells, and of dexamethasone suppression of vas- prednisolone, which include inhibition of lipid peroxidation and
cular permeability in the periphery and the brain. Despite this, stabilization of cell membranes. By reducing lipid peroxidation,
certain differences regarding dexamethasone effects were ob- the lazaroids reduce secondary tissue injury from ischemia and
served between the two models used for studying vascular per- trauma. In addition, arachidonic acid–induced vasogenic brain
meability. Suppression of permeability in the brain tumor edema has been inhibited in rats with the 21-aminosteroid
model is not as complete as in the periphery, and higher doses U74006F (14). Our results show that these non–GR-dependent
of dexamethasone are needed to be effective in the brain than effects are of marginal, if any, importance in tumor-associated
in the periphery. Although a dose of 0.2 mg/kg of dexametha- vascular permeability. The inability of non–GR-mediated ac-
sone suppressed permeability by z 90% in the skin, only 60% tions of glucocorticoids to suppress brain tumor–associated ce-
inhibition of permeability in the brain tumor was observed at a rebral edema is supported by the observations of Arbit et al.,
dose of 10 mg/kg. Possible explanations for these differences who found that the aminosteroid U-78517 actually increased
follow. (a) Permeability to large molecules (as measured by cerebral edema in rats with 9L gliosarcomas (29). In addition,
Evans blue extravasation in the skin) is more easily suppressed the aminosteroids lacked antipermeability effects in other rat
than permeability to small molecules (as measured by AIB in brain tumor models, the C6 astrocytoma (67) and the Walker
the brain). However, dexamethasone inhibition of permeabil- 256 carcinosarcoma (25), in which dexamethasone is effective.
ity is not complete in 9L brain tumor models even when Evans It is unknown why brain tumor–associated vascular permeabil-
blue is the tracer (54). (b) Dexamethasone is unable to inhibit ity is unaffected by non–GR-mediated steroids. Although the
hypoxic induction of VPF in 9L cells that may occur in vivo. 21-aminosteroids depend on free radical scavenging to reduce
However, 9L tumors implanted intracranially do not exhibit secondary brain injury after subarachnoid hemorrhage and
extensive necrosis (reference 22 and J.D. Heiss, unpublished trauma, oxygen free radicals are unlikely to be major causative
observations), which suggests that hypoxia may not be the pri- factors of peritumoral brain edema because superoxide dismu-
mary inducer of VPF in this model. (c) There may be perme- tase activity is similar in 9L brain tumors and normal brain

1406 Heiss et al.

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