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Endocrinology, doi:10.1210/en.2004-0179
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Endocrinology Vol. 145, No. 9 4320-4329
Copyright © 2004 by The Endocrine Society

Dual Hormonal Regulation of Endocrine Tissue Mass and Vasculature by Adrenocorticotropin in the Adrenal Cortex

Michael Thomas, Michelle Keramidas, Emmanuelle Monchaux and Jean-Jacques Feige

Institut National de la Santé et de la Recherche Médicale Equipe Mixte 105, Department of Cellular Responses and Dynamics, Commissariat à l’Energie Atomique, 38054 Grenoble, France

Address all correspondence and requests for reprints to: Dr. J.-J. Feige, Institut National de la Santé et de la Recherche Médicale Equipe Mixte 105, DRDC-ANGIO, Commissariat à l’Energie Atomique, 17 rue des Martyrs, 38054 Grenoble Cedex 9, France. E-mail: jjfeige{at}cea.fr.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The mass of healthy adult tissues is stable and their vasculature is quiescent, but this equilibrium is disrupted under certain physiological or pathological situations. There is an emerging concept indicating that these trophic changes may be initiated by modifications of the vasculature. In the current study, we documented over a period of 14 d the serial alterations occurring in both endocrine and endothelial compartments during adrenal atrophy induced by ACTH suppression in mice. After dexamethasone perfusion, a rapid fall of plasmatic ACTH and corticosterone concentrations was observed within the first 24 h. During the first 4 d of treatment, adrenal weight and adrenal cortex cellularity decreased rapidly. This was correlated with an inhibition of cell proliferation and a massive induction of endocrine cell apoptosis. Between d 4 and d 14, a slower but sustained decay of adrenal cortex size and cellularity was observed. This second phase was associated with progressive loss of vascular endothelial growth factor protein expression in the endocrine cells and regression of the vascular network. These data support the concept that ACTH controls adrenal cortex trophicity through a dual mechanism involving its antiapoptotic effect on endocrine cells and its indirect vascular endothelial growth factor-mediated action on endothelial cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE DEVELOPMENT OF a vascular network is a fundamental requirement for organ growth and differentiation during embryogenesis as well as during tissue repair or regeneration. The mass of adult tissues is essentially constant and results from a steady-state between apoptosis of senescent cells and tightly controlled regenerative proliferation. There are, however, a number of physiological and pathological situations under which this equilibrium can be disrupted. A few examples are the oestrus-dependent cycles of growth and vascular remodeling of the female endocrine organs (ovaries, uterus), the variations in fat tissue mass induced by modifications of the basal metabolism, and the regeneration of the liver after partial hepatectomy. In each of these situations, the vascular network appears to regress or develop in parallel with the differentiated cells of these organs, suggesting that some tightly controlled paracrine regulatory loops exist between these two cell types.

The adrenal cortex is a steroid hormone-producing tissue and one of the most highly vascularized tissues in the organism. The rapid release of corticosteroids into the blood flow is facilitated by a dense vascular network, which ensures that every adrenocortical cell is in contact with at least one endothelial cell (1). The pituitary hormone ACTH is the primary regulator of both fetal adrenal development and adult adrenal cortex trophicity. It is long known that hypophysectomy or specific deletion of corticotrophs from the pituitary results in the regression of the fasciculata zone of the adrenal cortex (2, 3). Inversely, perfusion of ACTH allows to regenerate the adrenal cortex to its original size and to restore corticosteroid secretion. Endocrinologists had explained this observation by a direct trophic effect of ACTH on adrenocortical endocrine cells. However, ACTH is not mitogenic for adrenocortical endocrine cells in vitro (in primary cultures) but rather inhibits proliferation through a cAMP-mediated process (4). The trophic effect of ACTH is thus more likely to be indirect and could be mediated by a cohort of paracrine factors acting through their effect on the vasculature. This hypothesis is supported by our recent in vitro observation that ACTH stimulates expression and secretion of vascular endothelial growth factor-A (named VEGF thereafter) by primary cultures of adrenocortical endocrine cells (5).

VEGF is the canonical member of a family of potent angiogenic factors (6). VEGF induces pleiotropic endothelial responses including stimulation of proliferation, differentiation, migration, survival, and tube formation (for review, see Refs.6, 7, 8). The biological effects of VEGF are mediated by two high-affinity tyrosine kinase receptors, VEGF-R1 (Flt-1) and VEGF-R2 (Flk-1/KDR) that are expressed at the surface of vascular endothelial cells and very few other cell types. Several studies have shown that interaction with VEGF-R2 is critically required to induce the full spectrum of VEGF biological responses, whereas VEGF-R1 rather seems to represent a decoy receptor, able to regulate in a negative fashion the activity of VEGF on the vascular endothelium by preventing VEGF binding to VEGF-R2 (6, 9). More recently, however, VEGF-R1 has been reported to induce some other endothelial responses in specific vascular beds, such as the induction of matrix metalloproteinase-9 in lung endothelial cells (10) or the release of hepatocyte growth factor and IL-6 by liver sinusoidal endothelial cells (11, 12).

A plausible paradigm for explaining the hormonal control of adrenocortical growth by ACTH is that VEGF (and possibly other angiogenic factors) is synthesized by endocrine cells under the control of ACTH and stimulates neovascularization (or prevents regression of the existing vascular network), which, in turn, controls endocrine tissue size. Although this is an attractive hypothesis, there is as yet few direct data to support it. To validate this concept in vivo, we developed an experimental mouse model to document alterations in the morphology, function, and proliferation of both endocrine and endothelial compartments after ACTH deprivation. Suppression of circulating ACTH was obtained within 24 h of chronic sc perfusion of dexamethasone (Dex) to 2-month-old male mice. Careful histochemical analysis of adrenal sections revealed two successive phases in the regression of the fasciculata/reticularis zone (ZF/ZR). Massive apoptosis of endocrine fasciculata cells was transiently observed during the first 4 d. It was followed by a strong and progressive decrease in VEGF protein expression between d 4 and 8 and a simultaneous degradation of the vascular network. These vascular alterations resulted in a second phase of tissue regression that stabilized between d 8 and 14. Taken together, our results indicate that adrenocortical atrophy induced by ACTH deprivation results from effects on both endocrine (increased apoptosis) and endothelial (vascular network regression due to decreased expression of VEGF by endocrine cells) compartments.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Experimental animals
All animal studies were approved by the institutional guidelines and the European Community for the Use of Experimental Animals. Two-month-old male OF-1 mice were obtained from Charles River Laboratories (Les Oncins, France) and maintained in the Animal Resources Center of our department. Dex was administered to mice at a dose of 1 mg/kg body weight per day by continuous infusion via sc implanted osmotic minipumps (Alzet Pump model 2002, Alzet, Cupertino, CA). Control mice were subjected to the same surgical procedure and implanted with osmotic minipumps filled with cyclodextrin, the Dex excipient. A group of mice was also implanted with two minipumps, one delivering 1 mg/kg·d Dex and one delivering 0.3 mg/kg·d human ACTH 1–39. The animals (n = 5 in each group) were killed at various time intervals after minipump implantation. The adrenal glands were collected carefully, cleaned from the surrounding adipose tissue, and weighed. One gland was either fixed overnight in 4% paraformaldehyde and embedded in paraffin or frozen in tissue-freezing medium (Jung, Nussloch, Germany). The other gland was directly dropped in denaturing solution and processed for RNA extraction.

RIAs
For assessment of circulating ACTH and corticosterone levels, blood was collected by cardiac puncture between 1400 and 1600 h. Plasma corticosterone concentrations were measured using an in-house RIA. The anticorticosterone antibody was a generous gift from Dr. B. Aupetit (Hôpital Pitié-Salpétrière, Paris, France). Plasma ACTH was measured using an immunoradiometric assay (Nichols Institute Diagnostics, San Juan Capistrano, CA).

Histology
The paraffin-embedded glands were entirely sectioned and mounted on slides coated with polysine. The middle section was stained with hematoxylin and eosin (H&E). H&E-stained sections from control, cyclodextrin-treated, or Dex-treated mice were examined under a light microscope (magnification, x100). The areas of the cortex and the ZF/ZR within the cortex were quantitated using Axiovision image analysis software (Zeiss, Le Pecq, France). Cell nuclei were counted within the ZF/ZR and expressed as number per high-power field (HPF).

Immunohistochemistry
For VEGF and 5-bromo-2'-deoxyuridine (BrdU) immunodetection, 5-µm-thick paraffin sections were deparaffinized, rehydrated, and microwaved in 10 mM citrate buffer (pH 6.0) at 800 W for either 2 x 5 min (VEGF) or 10 min and then 3 x 5 min (BrdU). Endogenous peroxidase activity was blocked by incubating sections with 1% H2O2 in methanol for 20 min. Slides were then incubated for 1 h with 0.5 µg/ml rabbit polyclonal antihuman VEGF antiserum (A20; Santa Cruz Biotechnology, Santa Cruz, CA), which recognizes all VEGF isoforms or with a rat anti-BrdU at a dilution of 1/75 (clone BU1-75; Harlan, Indianapolis, IN). After several washes, sections were sequentially incubated with biotinylated secondary antibodies and an avidin/biotinylated horseradish peroxidase complex (Dako A/S, Glostrup, Denmark). Peroxidase activity was revealed using diaminobenzidine tetrachloride as a chromogen (Dako A/S). Sections for VEGF detection were briefly counterstained with hematoxylin, and all were mounted.

For CD31 immunohistochemistry, frozen sections (8 µm) from fresh adrenal glands were fixed in 4% paraformaldehyde for 10 min. Sections were then washed 3 x 5 min in Tris-buffered saline containing 0.1% Tween 20, and endogenous peroxidases were blocked with 1% H2O2 in methanol for 20 min. Sections were then sequentially incubated for 1 h with a rat monoclonal anti-CD31 antibody (MEC 13.3; PharMingen, San Diego, CA; dilution 1:500) and for 45 min with a biotinylated rabbit antirat antibody (Dako). The final steps of the immunostaining were as described above.

Detection of apoptosis
Cells undergoing apoptosis were detected by in situ analysis of DNA strand breaks in histological sections using a nonradioactive fragment end labeling DNA fragmentation detection kit (Oncogene Research Products, Boston, MA) following the manufacturer’s recommendations. Sections incubated with biotinylated-deoxyuridine triphosphate in the absence of terminal dideoxy transferase confirmed the specificity of the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) detection (not shown).

Detection of lipid droplets
Frozen sections were fixed in 6% glutaraldehyde in PBS for 10 min, stained with Oil Red O (Sigma Chemical Co., St. Louis, MO) in isopropanol for 10 min and counterstained with hematoxylin. Oil Red O detects primarily cholesteryl ester droplets in steroidogenic tissues.

RNA isolation and semiquantitative RT-PCR
Total RNA was extracted from adrenal tissue in a 4 M guanidium isothiocyanate buffer using a rapid total RNA isolation system (RNAgents; Promega, Charbonnières, France).

Reverse transcription was performed on 1 µg total RNA with superScript II RnaseH reverse transcriptase (Invitrogen, Cergy Pontoise, France) under conditions recommended by the manufacturers. Before PCR, quantities of cDNA samples were adjusted to yield equal amplifications of mRNA encoding the housekeeping enzyme hypoxanthine phosphoribosyltransferase (HPRT). Specific oligonucleotide primers for HPRT; VEGF;VEGF-R1; VEGF-R2; neuropilin-1; melanocortin 2 receptor (MC2-R); scavenger receptor class B, type 1 (SR-B1); platelet endothelial cell adhesion molecule-1 (PECAM); and vascular endothelial (VE)-cadherin were designed based on published sequences (Table 1Go). According to the standardization, various amounts of cDNAs were used for each PCR. PCR were performed in a final volume of 25 µl containing 1x PCR buffer, 1.5 mM MgCl2, 200 µM deoxynucleotide triphosphates, 400 nM each primer, 0.5 U Taq polymerase (Q. Biogene, Montreal, Canada). The PCR conditions were: step 1, 94 C for 1 min; step 2, 25–35 cycles at hybridization temperature indicated in Table 1Go; and step 3, 72 C for 5 min. To ensure semiquantitative results in the RT-PCR assays, the number of PCR cycles for each set of primers was selected to be in the linear range of amplification. PCR products were visualized after electrophoresis on 1.5% agarose gels by ethidium bromide staining.


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  		TABLE 1. Primers used for semiquantitative and real-time RT-PCR

 
Real-time RT-PCR
VEGF, VEGF-R1, VEGF-R2, neuropilin-1, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression was quantitated by real-time RT-PCR on a LightCycler apparatus (Roche Diagnostics, Meylan, France). The PCR was performed using the primers indicated in Table 1Go and SYBR green PCR core reagents (LightCycler-FastStart Master SYBR Green I, Roche Diagnostics) according to the manufacturer’s instructions. PCR conditions were: step 1, 95 C for 10 min; step 2, 45 cycles consisting of 95 C for 15 sec, temperature indicated in Table 1Go for 5 sec (7 sec for VEGF-R1) and 72 C for 10 sec (14 sec for VEGF-R1). The results were normalized to GAPDH expression levels. The samples from at least three independent experiments were analyzed in duplicate. The data were analyzed using the RealQuant analysis software (Molecular Dynamics, Sunnyvale, CA).

Statistical analysis
All results are expressed as mean ± SD or ± SEM as indicated in the legends to the figures. Statistical comparisons were performed using ANOVA and Fisher’s protective least significant difference tests. Statistical significance was defined as P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Dex treatment induces adrenal cortex atrophy and suppresses corticosteroid secretion
We adapted to the mouse a protocol of Dex-induced adrenal atrophy that was initially described in the rat (13, 14). Various amounts of Dex were infused to 2-month-old male mice via a sc implanted osmotic minipump. Whereas higher doses generated side effects including liver and kidney infections, cachexia, and atony, infusion of 1 mg/kg·d Dex for 14 d induced atrophy of the adrenal glands without any apparent noxious effect on the animals. We first investigated the time course of changes in plasmatic hormone levels and adrenocortical tissue mass. As soon as 24 h after the onset of Dex delivery, plasma ACTH and corticosterone concentrations fell to extremely low levels and remained low for the entire duration of the experiment (Fig. 1Go, A and B). In control animals perfused with the Dex excipient, cyclodextrin, plasma ACTH, and corticosterone levels were similar to those of intact animals.



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  		FIG. 1. Rapid effect of Dex treatment on plasmatic ACTH and corticosterone concentrations. ACTH (A) and corticosterone (B) concentrations were measured by RIA in the plasma from mice treated with 1 mg/kg·d Dex for the indicated periods of time (diamonds) or with cyclodextrin for 14 d (squares). Each value is the mean ± SD obtained from five distinct mice. This experiment was repeated three times with similar results.

 
The time course of adrenal cortex regression was then analyzed. The weight of adrenal glands from Dex-treated mice was significantly reduced, compared with that of cyclodextrin-treated or control animals since d 4 of treatment (control, 5.62 ± 0.63 mg; cyclodextrin treated, 6.08 ± 1.32 mg vs. Dex treated, 3.70 ± 0.33 mg) (Fig. 2AGo). The adrenal weight of Dex-treated animals was maximally reduced (about 2-fold) after 8 d of treatment (2.98 ± 0.64 mg) and was not further modified after 14 d (3.04 ± 0.65 mg) (Fig. 2AGo). More interestingly, we evaluated the density of cells in the ZF/ZR of Dex-treated animals (measured as the number of positively stained nuclei per HPF). Three stages were observed: from d 0 to d 2, the density was unmodified; from d 2 to d 6, there was a rapid decrease in cell density; from d 6 to d 14, the decrease persisted but at a slower rate (Fig. 2BGo). The simultaneous histological observation of the regressing tissue revealed striking modifications. As shown in Fig. 3AGo, the surface of the cortex progressively decreased from d 2 until d 8 of Dex treatment. More delicate observation indicated that the size of the glomerulosa zone remained unchanged, whereas that of the fasciculata-reticularis zone was reduced down to 50% (after 8 d) and thus appeared as the main contribution to the observed adrenal cortex atrophy. As shown in Fig. 3BGo, besides the overall atrophy of the cortex, one could observe that the palissadic organization of the zona fasciculata was progressively lost and replaced by structures resembling acini. Moreover, the cytoplasm of fasciculata cells rapidly became clear and poorly stained by H&E. Lipid staining with Oil Red O allowed us to show that this was due to a massive accumulation of lipids in the steroidogenic cells of the ZF/ZR over time of Dex treatment (Fig. 3CGo). A cross-section through a control adrenal gland shows Oil Red O staining in the cortex but none in the medulla. Lipid droplets are abundant, small in size, and well defined throughout the cytoplasm of cells in the ZF/ZR but are much less abundant in zona glomerulosa (ZG) cells. In contrast, the size of the lipid deposits in the ZF/ZR increased by d 2, and they merged to fill the cytoplasm after 8 d of ACTH deprivation leading to hypertrophy of the cells.



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  		FIG. 2. Biphasic effect of Dex treatment on adrenal gland weight and cellularity. A, Mean weights ± SD of adrenal glands from mice treated with 1 mg/kg·d Dex for the indicated periods of time (diamonds) or with cyclodextrin for 14 d (squares). B, Cell count per HPF within the ZF/ZR of adrenals of untreated and Dex-treated mice. The individual dotted lines (marked 1 and 2) emphasize the distinct phases taking place during adrenal atrophy. Each value is the mean ± SD obtained from five distinct mice. This experiment was repeated twice with similar results. The statistical significance between untreated (D0) and Dex-treated mice was determined as described in Materials and Methods (*, P < 0.05).

 


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  		FIG. 3. Time course of histological and lipid content modifications of adrenal glands after Dex treatment. A, Low-magnification (x100) pictures of H&E-stained adrenal sections from mice treated for 0–14 d (D0 to D14) with 1 mg/kg·d Dex or for 14 d with cyclodextrin (CDx). The dotted lines mark the border between adrenal cortex and medulla. B, High-magnification (x400) pictures of H&E-stained adrenal sections (ZG/ZF) from mice treated with Dex for 0–14 d (D0 to D14). C, Frozen adrenal sections from mice treated with Dex for 0–14 d (D0 to D14) were stained with Oil Red O (magnification, x100). Data from one representative experiment are shown. Similar results were obtained in three independent experiments.

 
In an additional control experiment, we implanted several mice with two minipumps, one delivering the same concentration of Dex used in the aforementioned experiments and one delivering 0.3 mg/kg·d of ACTH 1–39. After 4 d of such a treatment, we observed that the weight and size of these animals’ adrenals were unmodified (data not shown), suggesting that the observed Dex effect was the consequence of ACTH suppression rather than a direct glucocorticoid effect.

Dex treatment induces down-regulation of proliferation and up-regulation of apoptosis in the remodeling adrenal cortex
Adrenal proliferation was studied during adrenal cortex regression by immunodetection of nuclear BrdU incorporation. BrdU was injected to mice 1 h before being killed. The first panel in Fig. 4AGo shows the typical distribution pattern of BrdU-positive cells in control or cyclodextrin-treated animals. Proliferating cells were predominantly located in the ZG and the external part of the ZF/ZR. Few positive cells were also detected in the deeper ZF/ZR. In Dex-treated animals, the number of positive cells decreased in the ZF/ZR by d 2, and the inner layer of zona fasciculata was devoid of proliferating cells (Fig. 4AGo). Careful microscopic observation allowed distinguishing flat endothelial cell nuclei from larger and round endocrine cell nuclei. We quantitated positive nuclei on a whole sagittal section that was carefully selected as one of the three central sections. The number of proliferating endocrine cells in the ZF/ZR decreased from 11 positive cells/section at d 0 to five at d 2 (55%) and two at d 8 (92%). In the endothelial cell compartment, the number of proliferating cells dropped from four positive cells/section at d 0 to one at d 2 (75%) and 0 at d 8 (100%). On d 8, most of the BrdU-positive cells were circumscribed to the ZG. In the adrenals of mice that were perfused simultaneously with Dex and ACTH for 4 d, the number of proliferating cells was slightly less than in control mice but clearly larger than in Dex-perfused mice.



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  		FIG. 4. Effect of Dex treatment on proliferation and apoptosis in the adrenal cortex. A, BrdU-positive nuclei from proliferating cells were visualized in adrenal gland sections from mice treated with 1 mg/kg·d Dex for 0–14 d (D0 to D14) or for 4 d with both Dex and 0.3 mg/kg·d ACTH (D4 + ACTH), as described in Materials and Methods. Brown dots indicate the presence of BrdU (magnification, x200). B, Detection of cell apoptosis by in situ analysis of DNA fragmentation in sections of adrenal glands from mice treated with Dex for 0–14 d (D0 to D14) (for details, see Materials and Methods). Apoptosis is indicated by dark staining (magnification, x200). The dotted lines delineate the border between adrenal cortex and medulla. Data from one representative experiment are shown. Similar results were obtained in three independent experiments.

 
Apoptosis was then detected on adjacent sections by TUNEL. As shown in Fig. 4BGo, whereas no apoptosis was detectable in control adrenals or in adrenals from animals treated for 8 or 14 d with Dex, a large number of TUNEL-positive cells was transiently observed on d 2 and 4 of Dex treatment. At these time points, TUNEL staining was restricted to the ZF/ZR. In contrast, in the adrenals of mice that were perfused simultaneously with Dex and ACTH for 4 d, no apoptotic cell was detectable.

Dex treatment induces inhibition of VEGF protein and mRNA expression and profound alterations of the adrenal cortex vasculature
To know whether the adrenocortical vasculature was also affected by Dex treatment, we next performed immunohistochemistry for CD31/PECAM, a specific marker for endothelial cells. As shown in Fig. 5AGo, CD 31 immunoreactivity was present throughout the adrenal cortex of control animals, revealing an extensive microvascular network. The capillaries in the ZF/ZR paralleled the columns of steroidogenic cells. After 4 d of Dex treatment, the capillaries appeared discontinuous and tortuous (Figs. 3BGo and 5AGo) with a shift of vessel diameters toward larger lumen as compared with cyclodextrin animals (Fig. 3BGo). Moreover, the lumen of those larger vessels was engorged with red blood cells, suggesting a stasis or a least a blood flow velocity reduction. No evidence of endothelial cell detachment or hemorrhagic foci was detected. At d 14, we noticed that these histological modifications became more profound and that the vascular density was markedly decreased. Interestingly, the vasculature of the ZG remained intact throughout the course of Dex treatment.



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  		FIG. 5. Effect of Dex treatment on adrenal cortex vasculature and VEGF expression. Sections of adrenal glands from mice treated with 1 mg/kg·d Dex for 0–14 d (D0 to D14) were immunostained for CD31 (A) or VEGF (B). The dotted lines indicate the border between adrenal cortex and medulla. Immunoreactivity is indicated by brown staining. Data from one representative experiment are shown. Similar results were obtained in three independent experiments.

 
The next step was to attempt to identify the factor that may trigger blood capillary remodeling during Dex treatment. Because VEGF is considered a prominent regulator of endothelial cell proliferation and survival and we previously observed that ACTH stimulates VEGF expression in primary cultures of adrenocortical endocrine cells (5), we wondered whether VEGF expression was also affected in vivo by changes in ACTH circulating levels. We therefore analyzed both VEGF protein and mRNA levels in the adrenals of Dex-treated mice. Immunohistochemical analysis of VEGF revealed a strong and uniform staining in the adrenal cortex of naive and cyclodextrin-treated mice. After Dex treatment, a progressive decrease in VEGF staining was observed, starting in the outer part of the ZF/ZR at d 2 and throughout the entire ZF/ZR after d 4. As shown in Fig. 5BGo, these changes were more pronounced at d 8 to reach an undetectable level by d 14. In contrast, VEGF immunoreactivity in the ZG did not change during the course of the treatment. The capsule and the medulla were negative for VEGF protein at all time points.

The expression level of the different VEGF mRNA isoforms was then analyzed by RT-PCR using a pair of primers that allowed the simultaneous amplification of distinct products specific for each of the four major VEGF isoforms. As shown in Fig. 6AGo, two major VEGF isoforms were expressed in mouse adrenal glands: VEGF120 and VEGF164. On d 2 of Dex treatment, the abundance of these isoforms was decreased, and this expression level then remained low for up to 14 d of treatment (Fig. 6AGo). Using real-time RT-PCR, the decrease of VEGF mRNA expression was quantitatively found to reach 40–60% on d 2–14 of Dex treatment (Fig. 6BGo). The magnitude of the reduction in the level of VEGF mRNA was significantly less than that seen for the protein (compare data in Fig. 5BGo with those in Fig. 6Go), which suggests that there is posttranscriptional or translational regulation of VEGF under these conditions.



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  		FIG. 6. Effects of Dex treatment on the expression of endocrine and endothelial cell markers. A, RT-PCR analysis of the expression of VEGF-A isoforms (the two bands correspond to specific amplification products of the VEGF120 and VEGF164 isoforms), VEGF-Rs (VEGF-R1/flt-1, VEGF-R2/flk-1, and neuropilin-1), endocrine cell markers (ACTH receptor/MC2-R, HDL receptor/SR-B1), and endothelial cell markers (PECAM and VE-cadherin) in the adrenal glands from mice treated with 1 mg/kg·d Dex for 0–14 d (D0 to D14). RT-PCR amplification of HPRT mRNA was used as an internal standard for the normalization of the samples. The results from one representative experiment are shown. Similar results were obtained in three independent experiments. B and C, Quantitative determination of adrenal VEGF-A and VEGF-Rs mRNA levels by real-time RT-PCR was performed as described in Materials and Methods. The ratio of level of expression of the gene of interest to that of GAPDH was normalized to 1 in control adrenals. The relative quantities of the following mRNAs were plotted as a function of time of Dex treatment: VEGF-A (B); VEGF-R1 (C, solid line), VEGF-R2 (C, continuous dotted line), and neuropilin-1 (C, discontinuous dotted line). Each value represents the mean ± SEM from 10 to 15 mouse adrenals collected in three independent experiments.

 
In parallel, we developed several specific RT-PCRs to measure the level of expression of VEGF receptors (VEGF-R1/flt-1; VEGF-R2/flk-1, and neuropilin-1) as well as markers of endocrine cells (the ACTH receptor MC2-R and the high-density lipoprotein (HDL) receptor SR-B1) and endothelial cells (PECAM and VE-cadherin) during the Dex treatment (Fig. 6AGo). Whereas the expression of VEGF-R1 and neuropilin did not appear to change during the treatment, that of VEGF-R2 was progressively reduced on d 4 and 8 and remained low on d 14. The inhibition reached 60% on d 14 as shown by the quantitative RT-PCR determinations in Fig. 6CGo. VE-cadherin and PECAM expressions decreased from d 4 up to d 8 and 14, whereas the expression of MC2-R and SR-B1 remained constant over time. It should be noted that the same amount of RNA isolated from the whole adrenal gland was used for these different RT-PCRs as controlled by the similar level of amplification of the mRNA encoding the housekeeping enzyme HPRT. Therefore, either a change in the ratio between endocrine and endothelial cells or the specific loss of expression of a given gene can result in a reduction of the amplification signal a cell type-specific gene. An overall regression of the adrenal gland, if not accompanied by a dedifferentiation, would not modify the amounts of RT-PCR amplification products.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Like other endocrine tissues, the adrenal cortex is composed of a complex admixture of steroidogenic cells, nerve cells, and smooth muscle and endothelial cells interacting to form a delicately organized network of blood capillaries that efficiently drain the corticosteroid secretions. This tissue is strictly dependent on the pituitary hormone ACTH for its development, growth, and adult maintenance. The mechanisms for this dependence are presently not fully elucidated. It has long been postulated that ACTH directly interacts with steroidogenic cells and controls their apoptosis and proliferation. The first description of apoptosis by Wyllie et al. (15, 16) in the 1970s was made in this tissue when these authors described the death of adrenocortical steroidogenic cells after ACTH deprivation or during the neonatal period. If the antiapoptotic effects of ACTH were largely confirmed (17, 18, 19), its proliferative action has remained more controversial. Although the extraordinary growth of the fetal adrenal cortex and the maintenance of the size of the adult cortex are dependent on an intact pituitary and intact ACTH secretion, it is also widely admitted that ACTH inhibits the proliferation of primary cultures of steroidogenic adrenocortical cells from various species (19, 20). If these steroidogenic cells are grown on extracellular matrix and in the presence of fibroblast growth factor-2, then they proliferate independently of the presence or absence of ACTH (21). The trophic effects of ACTH are thus likely to be mediated by the secretion of growth factors acting then in an autocrine or paracrine mode.

In the current study, we adapted to the mouse the well-established rat model of Dex-induced adrenal cortex regression and used it to demonstrate the contribution of the vasculature to the trophic effect of ACTH. This hypothesis was supported by our recent observation that the expression of VEGF, one of the most potent and specific angiogenic factors, is stimulated by ACTH in primary cultures of adrenocortical cells (5) and by the increasing number of reports showing that the mass of tissues such as prostate or fat is angiogenesis dependent (22, 23, 24, 25, 26, 27). We observed that infusion of Dex induced a dramatic breakdown of plasmatic ACTH and corticosterone levels within the first 24 h. The regression of the adrenal cortex was then progressively observed in a time range comprised between d 0 and d 14. It affected principally the ZF/ZR and was accompanied by two characteristic modifications: the density of cells in the remaining tissue decreased progressively over time and, simultaneously, the remaining cells became progressively engorged with lipid droplets. This latter process is likely to result from the suppression of steroidogenesis. Adrenocortical cells have four sources from which to draw cholesterol for corticosteroid synthesis: the HDL-selective uptake pathway, endogenous cholesteryl ester stores, the low-density lipoprotein receptor pathway, and de novo cholesterol synthesis from acetate. Under normal circumstances, HDL uptake is the most prominent source of cholesterol in rodent adrenocortical cells (28). We observed that the expression of the HDL receptor, SR-B1 (29, 30), was not modified during the course of Dex treatment. In the absence of ACTH, steroidogenesis is no longer stimulated and the expression of several steroidogenic enzymes is down-regulated but because steroidogenic cells still take up low-density lipoproteins, cholesterol accumulates in the cells.

Careful follow-up of adrenal weight and adrenocortical cell density revealed a similar biphasic phenomenon: a rapid decrease of the size and cellularity of the adrenal cortex during the first 4 d of Dex treatment followed by a more discrete decrease over the following days. This suggested that two distinct steps contributed to the regression. The first step appeared to involve inhibition of endocrine cell renewal and induction of endocrine cell apoptosis. During this initial period, the expression of VEGF slightly decreased, but the vascular network remained unmodified. After 4 d, apoptotic figures were no longer detectable, but the vascular network started to regress and the decrease in VEGF protein expression was more and more pronounced. According to these observations, ACTH controls the size and trophicity of the adrenal cortex through both an antiapoptotic effect on endocrine cells and a paracrine trophic effect on endothelial cells mediated by the ACTH-induced secretion of VEGF by endocrine cells. This regulation is more complex than the regulation of prostate tissue by androgens. Endothelial cells appear to undergo apoptosis 24 h sooner than epithelial cells during castration-induced ventral prostate involution. They are also first to proliferate during testosterone-stimulated prostate growth (24, 25, 31). Moreover, prostatic VEGF mRNA and protein levels are significantly reduced during castration and reinduced by testosterone, suggesting that this angiogenic factor could be the major mediator of these trophic effects.

Dex has been reported to induce apoptosis in a number of cell types through direct binding to the glucocorticoid receptors. However, the adrenal cortex does not seem to be sensitive to glucocorticoids because the adrenal glands from glucocorticoid receptor-deficient mice lack a central medulla but contain a normal cortex and synthesize corticosteroids (32). In accordance with these observations, we are confident that the effects of Dex described in the present study are likely to be due to the down-regulation of pituitary ACTH secretion because they were prevented by the simultaneous perfusion of ACTH 1–39.

The Dex-induced suppression of ACTH secretion by the pituitary had no effect on the abundance of MC2-R (ACTH receptor) mRNA. This is a somewhat surprising finding given previous reports that have suggested that, in vitro, the expression of MC2-R by ovine adrenocortical cells is regulated by glucocorticoids and ACTH (33, 34). However, Simmonds et al. (35) have shown that hypophysectomy of ovine fetuses did not alter the adrenocortical expression of MC2-R mRNA. Conversely, it has been shown that ACTH infusion to sheep fetus is able to increase cortisol synthesis in the adrenal gland without up-regulation of MC2-R mRNA (36). However, in a recent study (37), treatment of mice for 6 d with a dose of Dex 5 times larger than the one used in the present work appeared to induce a 60% decline in adrenocortical MC2-R mRNA levels. Interestingly, in this study, MC2-R mRNA levels were not restored by daily ACTH injections for 7 d, whereas in our hands, normal plasmatic ACTH and cortisol levels were restored within 24 h after cessation of Dex perfusion (data not shown). These differences, combined with our personal observations, indicate that high doses of Dex induce several noxious effects on mice and that the dose of Dex used in these studies has to be minimal to observe clean ACTH-related effects.

Dex-induced down-regulation of VEGF expression is unlikely to result from a direct effect through the glucocorticoid receptor because glucocorticoids do not modify VEGF expression in primary cultures of bovine adrenocortical cells (our unpublished observations), and the expression of glucocorticoid receptors in adrenocortical endocrine cells has never been established. Although in some cell types, Dex decreases VEGF expression (38, 39), we can rule out such a direct effect in adrenocortical cells.

Mouse VEGF gene undergoes differential splicing to generate four variants encoding the isoforms VEGF120, VEGF144, VEGF164, and VEGF188. In mouse, human, and bovine adrenocortical cells, VEGF120 and VEGF164 are the most abundantly expressed isoforms (5, 37, 40). Here we demonstrate that ACTH deprivation alters the expression of these two major variants that are primarily secreted and exert mitogenic and permeabilizing effects on endothelial cells (7). The decline in these two mRNA isoforms in our studies is correlated with a decline in VEGF protein. However, immunohistochemical detection of VEGF in the regressing cortex revealed an almost complete suppression of the protein (that we were unfortunately unable to quantitate by alternative methods such as Western blotting due to the lack of sensitivity of commercially available antibodies), whereas VEGF mRNA decreased by only 30–40% as quantitatively measured by real time RT-PCR. This definitely suggests that ACTH regulates VEGF translation as well as transcription or mRNA stability. The control of VEGF expression is a highly complex and coordinated process. Besides the transcription factors that bind VEGF promoter and control its transcription (6), several proteins that bind to VEGF mRNA 3'-untranslated region and promote its stabilization (e.g. HuR) or destabilization [e.g., tetradecanoyl pherbol acetate (TPA)-inducible sequence 11b (TIS11b)] have been characterized (41, 42). In addition, VEGF mRNA translation is also highly regulated with cap-dependent and internal ribosome entry site (IRES)-dependent initiation processes. Therefore, a more detailed analysis is definitely required to understand the different levels at which ACTH might intervene to induce VEGF expression.

Consistent with a proposed paracrine role for VEGF in tissues, we previously observed that expression of VEGF-R1 and VEGF-R2 is restricted to endothelial cells of the adrenal cortex (43). Dex treatment selectively down-regulated VEGF-R2 expression without altering VEGF-R1 expression. Because VEGF-R2 is now considered the major signal-transducing tyrosine kinase receptor for VEGF, whereas VEGF-R1 is either a decoy or a coreceptor (6, 44), this down-regulation is likely to cause the observed alterations of the vascular network that develop during the same time frame, i.e. between d 4 and d 14. Because the decrease in VEGF mRNA and protein levels slightly precedes by a couple of days that of VEGF-R2, it is tempting to speculate that VEGF expression levels in endocrine cells might control VEGF-R2 expression levels in endothelial cells. For those who might not be convinced that such a small decrease in VEGF mRNA levels might be responsible for such dramatic effect on adrenocortical size and vascularization, it should be recalled that a 50% reduction in VEGF gene dosage, as observed in heterozygous knockout mice, was sufficient to induce the complete phenotype of homozygous knockout mice, i.e. embryonic death at d 10.5 due to abnormal yolk sac and embryo proper vascularization (45, 46).

We used two markers of endothelial cells, namely PECAM and VE-cadherin, to check the relative amount of endothelial cell-derived mRNA within the RNAs prepared from whole adrenals as a function of regression time. Interestingly, PECAM expression only slightly decreased by d 14, probably reflecting the massive loss of blood capillaries observed at this time point, whereas the VE-cadherin expression was more strongly decreased, suggesting that, like VEGF-R2, this gene might be indirectly regulated by VEGF levels.

In conclusion, we have shown that ACTH, in addition to its known function as a regulator of adrenocortical steroidogenesis and trophicity, is also required for the maintenance of adult mouse adrenal cortex vasculature. We have presented evidence suggesting that the remodeling observed after ACTH deprivation is caused by coordinated changes of the steroidogenic and endothelial compartments. Unlike in the prostate, regression of the vasculature caused by decreased VEGF expression appears to be a secondary effect of ACTH deprivation. It is, however, significant and contributes to about 50% of the overall regression of the adrenal cortex tissue caused by in vivo Dex treatment. Additional experiments are now ongoing in our laboratory to examine whether antiangiogenic drugs might compromise regeneration of the adrenal cortex that is observed within 6 d after cessation of Dex treatment.


   Acknowledgments
 
We are indebted to M. Martinie and Dr. P. Faure (Department of Integrative Biology, University Hospital of Grenoble) for their significant help in the measurement of plasmatic ACTH.


   Footnotes
 
This work was supported by Institut National de la Santé et de la Recherche Médicale (Equipe Mixte 105), Commissariat à l’Energie Atomique (Department of Cellular Responses and Dynamics), and the Association pour la Recherche sur le Cancer (Equipment Grant 7626 and Research Grant 4713) (to M.T.).

M.T. and M.K. were equal contributors to this article.

Present address for E.M.: Département de Génie Chimique, Université de Sherbrooke, 2500 Boulevard Université, Sherbrooke, Québec, Canada J1K 2R1.

Abbreviations: BrdU, 5-Bromo-2'-deoxyuridine; Dex, dexamethasone; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HDL, high-density lipoprotein; H&E, hematoxylin and eosin; HPF, high-power field; HPRT, hypoxanthine phosphoribosyltransferase; MC2-R, melanocortin 2 receptor; PECAM, platelet endothelial cell adhesion molecule-1; TUNEL, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling; VE, vascular endothelial; VEGF, vascular endothelial growth factor-A; VEGF-R, VEGF receptor; ZF/ZR, fasciculata/reticularis zone; ZG, zona glomerulosa.

Received February 11, 2004.

Accepted for publication May 27, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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