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Original Articles |
Reproductive Endocrinology Center, Department of Obstetrics, Gynecology, and Reproductive Sciences (J.L.S., S.M., R.N.T., R.B.J.), University of California, San Francisco, California 94143-0556; and the Department of Cardiovascular Research (N.F.), Genentech, Inc., South San Francisco, California 94080
Address all correspondence and requests for reprints to: Robert B. Jaffe, M.D., Reproductive Endocrinology Center, Box 0556, University of California, San Francisco, California 94143-0556. E-mail: robert_jaffe{at}quickmail.ucsf.edu
Abstract
The human adrenal cortex has a complex vasculature that is essential for growth, organ maintenance, and access of secreted hormones to the circulation. Growth and function of the adrenal cortex are regulated by corticotropin (ACTH), the actions of which are in part mediated by locally produced growth factors. As cortical growth and vascularization must increase in a coordinated manner, we hypothesized that ACTH also influences adrenal cortical angiogenesis by stimulating the local expression of specific angiogenic factors. Vascular endothelial growth factor (VEGF) is a potent endothelial cell-specific angiogenic peptide, the expression of which has been detected in adrenal cortical cells. Therefore, we examined the localization of VEGF expression in the midgestation (16–20 weeks) human fetal adrenal cortex and determined whether VEGF expression and secretion by isolated human fetal adrenal cortical cells are regulated by ACTH. By immunohistochemical analysis, strong cytoplasmic staining for VEGF was detected in scattered clusters of fetal zone (inner cortical compartment) cells. In contrast, cells in the outer, definitive zone of the cortex stained only weakly for VEGF. The predominant staining for VEGF in the fetal zone correlated with the extensive vasculature of this zone as detected by immunohistochemical staining for von Willebrand factor, which is specific for endothelial cells. In primary cultures of human fetal adrenal cortical cells, ACTH (1 nmol/L) and forskolin (10 µmol/L) increased the abundance of messenger ribonucleic acid transcripts encoding VEGF, as assessed by Northern and slot blot analyses. The stimulatory effect of ACTH and forskolin on VEGF gene expression occurred within 2 h of agonist exposure and persisted for at least 24 h. ACTH and forskolin also increased VEGF protein secretion by fetal adrenal cortical cells, as assessed by enzyme-linked immunosorbent assay for VEGF in fetal adrenal cortical cell-conditioned medium. A significant (P < 0.05) increase in VEGF secretion was detected as early as 8 h after ACTH or forskolin treatment. By 24 h after the addition of ACTH or forskolin, VEGF secreted from isolated human fetal adrenal cells was increased 5- to 6-fold. These data demonstrate that the human fetal adrenal cortex, particularly the cells of the inner fetal zone, express VEGF and that VEGF expression and secretion by these cells are directly regulated by ACTH and the activation of adenylate cyclase. Thus, VEGF may be a local regulator of adrenal cortical angiogenesis and an important mediator of the tropic action of ACTH, ensuring the coordination of ACTH-stimulated cortical growth and vascularization.
THE ADRENAL cortex is a dynamic endocrine organ that exhibits remarkable plasticity depending upon the extent of tropic hormone exposure. Excessive production of corticotropin (ACTH) by the pituitary (e.g. Cushing’s syndrome) promotes adrenal cortical hypertrophy, whereas the lack of ACTH results in cortical atrophy. During human fetal life, the adrenals are enlarged, primarily because of the unique cortical compartment known as the fetal zone (for review, see Ref.1). By midgestation, fetal adrenals are as large as their associated kidneys, and the fetal zone occupies 80–90% of the cortical volume (2). Growth and function of the human fetal adrenal cortex are regulated by ACTH secreted by the fetal pituitary (3, 4, 5, 6), the growth stimulatory actions of which are mediated in part by locally produced growth factors acting in an autocrine and/or paracrine manner (7, 8).
As with other endocrine organs, the adrenal cortex requires a vasculature to facilitate access of hormone products to the circulation. In humans, the adrenal vasculature is established by the eighth week of gestation when the adrenal is supplied by arteries from the descending aorta, and the capillary sinusoids within the gland form a continuum with the general circulation (9). Later in gestation, venous drainage develops with formation of the central adrenal vein (10), and a centripetal vascular flow pattern develops (11). The factors responsible for regulating the development of this complex adrenal vasculature are not known. However, it is clear that regulation of the vasculature must be coordinated with tropic regulation of the cortex, so that blood vessel formation and cortical growth are synchronized. Therefore, we hypothesized that ACTH-stimulated adrenal cortical vacularization is mediated by specific angiogenic factors.
One factor that may be involved in the regulation of adrenal vascularization is vascular endothelial growth factor (VEGF), a potent angiogenic glycoprotein, the actions of which are limited to vascular endothelial cells. VEGF stimulates the full cascade of events required for blood vessel formation (12) and also increases vascular permeability (13, 14). The expression of VEGF has been detected by in situ hybridization in the adult guinea pig (15) and adult rat (16) adrenal cortex. In the human fetal adrenal cortex, we found that VEGF was present in scattered cortical cells (17). Based on these data, we hypothesized that VEGF regulates the development and maintenance of the human fetal adrenal vasculature. As the regulation of VEGF expression by human fetal adrenal cortical cells has not been examined, and tropic hormone regulation of adrenal VEGF expression would be an effective mechanism for coordinating angiogenesis and cortical growth, we examined whether ACTH regulates VEGF expression by human fetal adrenal cortical cells.
Materials and Methods
Materials
Human fetal adrenal glands were obtained from second trimester fetuses (16–20 weeks; gestation estimated by foot length) after elective termination of pregnancy by dilatation and evacuation. Glands were collected immediately after pregnancy termination and placed in ice-cold tissue culture medium (see below). The study protocol was approved by the committee on human research of the University of California-San Francisco (UCSF).
Human ACTH [ACTH-(1–24); Cortrosyn] was obtained from Organon (West
Orange, NJ). Forskolin was obtained from Sigma Chemical Co. (St. Louis,
MO). A rabbit polyclonal antibody to human VEGF was obtained from Santa
Cruz Biotechnology (Santa Cruz, CA), and an antibody to human von
Willebrand factor (vWF) was obtained from Dako (Santa Barbara, CA). The
full-length complementary DNA (cDNA) encoding the steroidogenic enzyme
cytochrome P450 17
-hydroxylase, 17,20-lyase (P450c17) was provided
by Dr. W. L. Miller, UCSF (18). The full-length cDNA encoding
human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was obtained
from American Type Culture Collection (Rockville, MD).
Cell culture
Primary cultures of midgestation human fetal adrenal cortical cells were prepared as previously described (19). Briefly, the adrenal capsule was dissected away from the underlying cortical tissue, and the remaining fetal zone was dispersed by enzymatic digestion with collagenase. The dispersed cells were centrifuged and resuspended in culture medium, consisting of DMEM H-16/Ham’s F-12 (1:1) supplemented with 10% FCS, 2 mmol/L glutamine, and 50 µg/mL gentamicin (Cell Culture Facility, UCSF). Cells were plated onto culture plates (Falcon, Los Angeles, CA) or eight-chamber slides (Lab-Tek, Nunc, Naperville, IL) at a density of approximately 10,000 cells/cm2 and incubated in a humidified environment at 37 C in 95% air-5% CO2. Media were changed every 48 h. At the initiation of experiments (usually 96 h after plating), the concentration of FCS was reduced to 2%, and 1 nmol/L ACTH or 10 µmol/L forskolin were added to the cells and incubated for 1–24 h. These concentrations of ACTH and forskolin were chosen because they induce near-maximal stimulation of steroidogenesis (7, 8). Media were then collected and stored at -20 C for subsequent assay of VEGF, and the remaining cells were processed for total ribonucleic acid (RNA) isolation.
RNA analysis
Northern blot and slot blot analyses were used to assay the abundance of specific messenger RNA (mRNA) transcripts. Total RNA was extracted and purified from cultured cells using the method of Chomczynski and Sacchi (20). For Northern blot analysis, total RNA (5–10 µg) was denatured in 2.2 mol/L formaldehyde at 60 C for 15 min, subjected to electrophoresis through 1.2% agarose-formaldehyde gels, and transferred to nitrocellulose membranes (Nytran, Schleicher and Schuell, Keene, NH) by capillary action. For slot blot analysis, total RNA (5–10 µg) was denatured in 50% formamide-7% formaldehyde-1 x SSC (0.15 mol/L NaCl and 0.015 mol/L Na citrate) at 68 C for 20 min and then applied to nitrocellulose membranes by vacuum using a slot blot manifold (Schleicher and Schuell). In both cases, the RNA was cross-linked to the membranes by UV irradiation (Stratalinker, Stratagene, La Jolla, CA) and prehybridized at 68 C in hybridization buffer (Quickhyb, Stratagene) for 15 min. All probes were synthesized by random primer extension in the presence of [32P]deoxy-CTP to a specific activity of 1–2 x 109 dpm/µg. For VEGF, a 921-bp fragment corresponding to nucleotides 335-1256 of human VEGF was used as a template (21). The human VEGF cDNA fragment was designed to span the protein-coding region of the gene and recognize all known VEGF variants. The labeled cDNA was denatured and hybridized to membranes for 2 h at 68 C. Membranes were then subjected to stringency washes with 2 x SSC-0.1% SDS (15 min at room temperature) and 0.1 x SSC-0.1% SDS (20 min at room temperature, then 20 min at 65 C) and exposed to Kodak XAR-5 film (Eastman Kodak, Rochester, NY) at -80 C with intensifying screens for 2–4 days. The molecular sizes of the RNA species detected by Northern blot analysis were calculated by extrapolation from the 18S and 28S ribosomal bands. Relative abundance of mRNA transcripts was estimated by computer-assisted densitometry (Bio Image, Ann Arbor, MI). All data were normalized to the abundance of transcripts encoding GAPDH, which was constitutively expressed. Probes were removed by washing the membranes in distilled water at 100 C. Complete removal of probe was confirmed by autoradiography before reprobing.
Immunocytochemistry
VEGF protein was localized by immunocytochemistry in human fetal adrenal cells cultured on chamber slides after fixation in 4% paraformaldehyde and in paraffin sections (10 µm) of human fetal adrenal glands. Sections of whole glands also were analyzed for vWF, an endothelial cell-specific marker. A modified avidin-biotin-peroxidase method was used, as previously described (17). Slides and sections were incubated overnight with antibody to VEGF (10 µg/mL) or vWF (14 µg/mL) at 4 C in a humidified chamber. Normal rabbit serum at the same protein concentrations was used as a control. Sections were then washed with Tris-buffered saline and incubated with an avidin-peroxidase complex (Vector Laboratories, Burlingame, CA), and signals were detected by substrate reaction with 0.05% diaminobenzidine and 0.001% H2O2. The sections were lightly counterstained with hematoxylin. Cells grown on chamber slides were not counterstained.
Enzyme-linked immunosorbent assay (ELISA)
Concentration of VEGF in human fetal adrenal cortical
cell-conditioned medium was assayed by ELISA, as previously described
(22). Before assay, medium samples were concentrated approximately
5-fold using Centricon-10 apparati (Millipore, Bedford, MA). Standards
(0.03–2 ng/mL recombinant VEGF165) and 3-fold serially
diluted samples in PBS containing 0.5% BSA, 0.05% polysorbate 20,
0.25% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate
(CHAPS), 0.2% bovine 
-globulin (Sigma Chemical Co., St. Louis, MO),
5 mmol/L ethylenediamine tetraacetate, and additional 0.35 mol/L NaCl
were incubated on the plate for 2 h. Bound VEGF was detected using
biotinylated monoclonal antibody to VEGF (mAb 4.6.1), followed by
streptavidin peroxidase (Sigma) and 3,3',5,5'-tetramethyl benzidine as
the substrate. Absorbance was read at 450 nm on a plate reader
(Molecular Devices, Menlo Park, CA). The standard curve was fitted
using a four-parameter nonlinear regression curve-fitting program. Data
points that fell in the linear range of the standard curve were used
for calculating the VEGF concentration in the samples. The assay was
linear for VEGF in conditioned medium and was sensitive to 0.2
ng/mL.
Statistical analysis
All experiments were repeated a minimum of three times. Time-course data were analyzed by repeated measures ANOVA, using Fisher’s correction for multiple comparisons. Significant differences were accepted when two-tailed analysis yielded P < 0.05.
Results
Localization of VEGF and vWF
Localization of VEGF protein in midgestation human fetal adrenal
sections was determined by immunocytochemical staining (Fig. 1
). The highest abundance of VEGF protein
was detected in fetal zone cells. Specific staining for VEGF in fetal
zone cells was mainly cytoplasmic, and its intensity varied among
cells. Cells in the definitive zone (narrow band of tightly packed
cells surrounding the fetal zone) exhibited only weak staining for VEGF
peptide. Immunocytochemical staining for vWF, which is specific for
endothelial cells, revealed an extensive vascular network throughout
the fetal adrenal cortex and capsule in midtrimester human fetal
adrenal glands (Fig. 1). Distinct blood vessels were detected
traversing the definitive zone from the capsule toward the center of
the gland. The fetal zone vasculature consisted of numerous sinuses
lined with endothelial cells.
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To determine whether ACTH regulates VEGF gene expression, human
fetal adrenal cortical cells were treated for 1–24 h with ACTH (1
nmol/L). Total RNA was then analyzed by either Northern or slot blot
analysis. ACTH increased the steady state abundance of mRNA encoding
VEGF (Fig. 2A). Transcripts of
approximately 4.4 and 4.2 kilobases (kb) were detected, which may
represent differential splicing of VEGF mRNA. Data from all experiments
(n = 4) were normalized to GAPDH and are summarized in Fig. 2B.
Changes in VEGF mRNA abundance in response to ACTH with time were not
statistically significant (P = 0.19, by repeated
measures ANOVA) due to the high variability between experiments;
however, in general, an increase in VEGF mRNA was first observed 2–4 h
after the addition of ACTH. In most experiments, the maximum levels of
VEGF mRNA were detected 4 h after ACTH addition and persisted for
up to 24 h. As indicated below, ACTH treatment resulted in a
significant increase in VEGF peptide secretion by human fetal adrenal
cortical cells. Forskolin (10 µmol/L) mimicked the effects of ACTH on
the accumulation of mRNA encoding VEGF (Fig. 2C). As expected, ACTH and
forskolin both increased the abundance of mRNA encoding P450c17.
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, middle
panels). As expected, the adrenal cortical cells retracted in
response to ACTH and forskolin, confirming the biological activity of
these agonists. Staining was close to background levels in
ACTH-stimulated cells incubated with normal rabbit serum or when
immunoabsorbed primary antiserum was used (data not shown)
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The effect of ACTH (1 nmol/L) and forskolin (10 µmol/L) on VEGF
secretion by human fetal adrenal cortical cells into the culture medium
was determined by ELISA (n = 3). ACTH and forskolin increased VEGF
secretion by cultured human fetal adrenal cortical cells (Fig. 4). Twenty-four hours after ACTH
addition, mean VEGF levels in the medium were increased 5.6-fold
compared with control values. A significant (P < 0.05)
increase in conditioned medium VEGF was detected as early as 8 h
after ACTH addition. The effects of ACTH on VEGF secretion by cultured
human fetal adrenal cortical cells were mimicked by forskolin.
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The endocrine function of the adrenal cortex requires an extensive vasculature through which secreted hormones gain access to the circulation. Clearly, regulation of adrenal vascularization and growth must be coordinated to ensure that the cortical mass has appropriate vascular support essential for both growth of the adrenal cortex and its endocrine function. ACTH is the principal regulator of adrenal cortical growth and function. The tropic actions of ACTH are mediated in part by a cohort of locally produced growth factors (7, 8). As ACTH is not an angiogenic factor per se, it is likely that its effects on adrenal cortical vascularization also are mediated by specific angiogenic factors, one of which may be VEGF. Previously, we showed that the human fetal adrenal cortex expresses VEGF (17). In the present study we assessed the localization of VEGF in the midgestation human fetal adrenal cortex and determined whether its expression and secretion by fetal adrenal cortical cells are regulated by ACTH. Our data show that 1) the human fetal adrenal cortex is highly vascular (based on the immunocytochemical localization of vWF), which is consistent with its function as an endocrine organ; 2) the human fetal adrenal cortex expresses the potent angiogenic factor, VEGF, which may regulate cortical vascular development; and 3) VEGF expression and secretion by human fetal adrenal cortical cells are up-regulated by ACTH and factors that augment intracellular cAMP production. Taken together, these findings suggest that VEGF plays a role in the vascular development of the human adrenal cortex and that it may be one of a cohort of locally expressed growth factors that mediate the actions of ACTH.
We found that the fetal zone is the principal site of VEGF synthesis. This pattern of VEGF localization is consistent with the fetal zone being the most vascular compartment in the cortex and the primary site of adrenal cortical growth. Thus, VEGF may act as a local regulator of fetal zone vascularization. The fetal zone vasculature comprises an extensive sinusoidal plexus. In contrast, the vasculature of the definitive zone is composed of distinct arterioles that arise from terminal branches of the capsular arterial network and enter the gland along connective tissue trabeculae. Therefore, as the cortex grows, the bulk of neovascularization would be expected to occur in the fetal zone. This vascular arrangement results in centripetal blood flow from the capsule through the definitive zone and into the sinusoidal network of the fetal zone to eventually drain into the central vein.
The expression and secretion of VEGF by human fetal adrenal cortical cells were increased by ACTH and forskolin. The almost identical effects of ACTH and forskolin are consistent with the actions of ACTH being mediated through the activation of adenylate cyclase and the subsequent increase in intracellular cAMP. Other actions of ACTH, including the induction of steroidogenic enzyme (e.g. P450c17) and basic fibroblast growth factor (7) expression, also are mediated through G protein-coupled activation of adenylate cyclase, indicating that the expression of VEGF is part of a complex series of events triggered by ACTH stimulation. VEGF transcripts approximately 4.4 and 4.2 kb in size were detected, which probably represent differential mRNA splicing. We observed a similar pattern of VEGF transcripts in other cell types (17, 22). Although the increased abundance of VEGF mRNA in response to ACTH was variable and not statistically significant, it was evident as early as 2 h after the addition of ACTH and correlated with a statistically significant increase in VEGF peptide secretion. The rapidity of this response is consistent with the direct regulation of VEGF gene expression and peptide secretion by ACTH. Thus, these studies show that ACTH may coordinate adrenal cortical growth and vascularization by increasing the expression of local growth and angiogenic factors, including VEGF.
Acknowledgments
We thank Janet Lee, Y. Gloria Meng, Evelyn Garrett, Shy Tassa, Kit Garcia, Naina Singh, and Dr. Wai Lee Wong for their assistance with this study. We also thank Dr. Jin Kim and Ji Lu for providing monoclonal antibodies to VEGF.
Footnotes
1 This work was supported in part by NIH Grants HD-08478 and
P30–11979. Presented in part at the 43rd Annual Meeting of the Society
for Gynecological Investigation, Philadelphia, PA, March 1996. 
2 Present address: Vincent Memorial Obstetrics and Gynecology
Service, Massachusetts General Hospital, Boston, Massachusetts
02114.
Received October 3, 1997.
Revised December 23, 1997.
Accepted December 31, 1997.
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