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Endocrinology, doi:10.1210/en.2003-0099
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Endocrinology Vol. 144, No. 10 4607-4615
Copyright © 2003 by The Endocrine Society

Expressional Regulation of the Angiopoietin-1 and -2 and the Endothelial-Specific Receptor Tyrosine Kinase Tie2 in Adrenal Atrophy: A Study of Adrenocorticotropin-Induced Repair

Olivier Féraud, Christine Mallet and Isabelle Vilgrain

Equipe Mixte Institut National de la Santé et de la Recherche Médicale (INSERM) (EMI 02-19), Laboratoire de Développement et Vieillissement de L’Endothélium (O.F., I.V.); and INSERM EMI 01-05 (C.M.), Département Réponse Dynamique Cellulaire, Commissariat à l’Energie Atomique, 38054 Grenoble Cedex 9, France

Address all correspondence and requests for reprints to: Dr. Isabelle Vilgrain, Commissariat à l’Energie Atomique, Grenoble, Département Recherche Dynamique Cellulaire, INSERM EMI 02-19, Laboratoire de Développement et Vieillissement de l’Endothélium, 17 rue des Martyrs, 38054 Grenoble Cedex 9, France. E-mail: ivilgrain{at}cea.fr.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Angiopoietin-1 (Ang-1), a newly discovered ligand of the endothelial-specific tyrosine kinase receptor Tie-2, has been found to promote cell survival, vascular maturation, and stabilization, and to function in concert with vascular endothelial growth factor. Adrenal gland has an intense capillary network that regulation remains to be documented. Recently, we demonstrated that vascular endothelial growth factor, and its receptors are expressed in mouse adrenal in vivo, but no detailed study on Ang expression in the adrenal has been reported. The present study shows the expression of Tie2 receptors, Ang-1, and its endogenous antagonist, Ang-2 in mouse adrenal in vivo. Immunohistochemistry disclosed that Tie2 colocalized with platelet-endothelial-cell-adhesion-molecule in endothelial cells from normal mouse adrenal. Daily administration of dexamethasone (DEX) (0.5 mg/100 g body weight·d) for 6 d in mice, decreased steroidogenic function of adrenal as shown by inhibition of the 36-kDa ACTH receptor protein expression, and decreased plasma corticosterone level [control from 465 ± 35 ng/ml to 114 ± 18 ng/ml in DEX group (P < 0.001)]. Using semiquantitative RT-PCR, we demonstrate that DEX treatment down regulates Ang-1 mRNA levels by 3- to 4-fold. No significant changes in Ang-2 were detected between control and DEX groups, resulting in an altered Ang-2 to Ang-1 relative ratio. The Tie2 receptor was also found to be down-regulated in DEX group at both mRNA and protein level. ACTH was found to play a causal role in DEX-induced decrease in Ang-1/Tie2 system, because 7 d treatment with long acting 1–39 ACTH (30 IU/kg·d) increased Ang-1, Tie2 expression, and plasma corticosterone back to control levels. These results reinforce the role of ACTH in the regulation of angiogenic factors in adrenal gland and suggest that the Ang/Tie2 system might represent a key player for stabilization of adrenal endothelium.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
EARLY VASCULAR DEVELOPMENT (vasculogenesis) comprises complex regulated processes such as proliferation, differentiation, and migration of endothelial cells that finally coalesce to form a primitive vascular network (1). Subsequently, the immature, primitive network undergoes remodeling processes involving sprouting, branching, differential growth of vessels, and recruitment of supporting cells (angiogenesis) to form a mature cardiovascular system (1) Once differentiated, the mature vasculature remains relatively stable. Central for regulation of these processes are two families of growth factors, the vascular endothelial growth factor (VEGF) and the recently discovered Ang (i.e. Ang-1 through -4) (2, 3, 4, 5), which are essentially involved in maturation, stabilization, and remodeling of vessels. Ang action is targeted toward the vascular endothelium, because both of their receptors, (i.e. tyrosine kinase with Ig and epidermal growth factor homology) (Tie)-1 and Tie2 are specifically expressed in vascular endothelial cells (6, 7). The four identified Ang use the associated receptor tyrosine kinase Tie2. Although a functional ligand for the related Tie-1 receptor remains unidentified, it is well known that Tie-1 functions in controlling vascular endothelial cell integrity. Hence, embryos deficient in Tie-1 die because of the resulting edema and localized hemorrhage (8, 9). In vivo studies revealed a close interaction of the Ang-1/Tie2 system with VEGF for vascular development. In Ang-1- or Tie-2-deficient embryos, a VEGF-mediated primitive vasculature develops, but embryos fail to remodel and stabilize the preformed vasculature, which finally leads to embryonic death (4, 6, 9). Transgenic overexpression of Ang-1 in the skin of mice leads to more numerous dermal capillaries and venules that are highly branched and characterized by increased diameters. However, the vasculature displays structural integrity and functionality (10). In contrast to skin-targeted VEGF expression that causes hyperpermeability and leakiness of vessels, Ang-1 overexpression results in a nonleaky vasculature, even under induced inflammatory conditions (11). In line with a potentially stabilizing role of Ang-1 for mature vessels, Ang-1 is found to be constitutively expressed in the adult (3). By contrast, Ang-2, representing a natural Tie2 antagonist, is highly induced at sites of vascular remodeling in the adult, such as the female reproductive tract (3), or during tumor growth and metastasis (12), suggesting that Ang-2 mediates a destabilization of existing vessels leading to a more plastic state. This situation then converts to active vascular remodeling in the presence of VEGF or to regression of frank vessels in the absence of VEGF (3, 12). Thus, the Ang appear to play a key role in coordinating the growth of new blood vessels by sending permissive signals via Tie2 to either promote vascular maturation/integrity, or destabilize vessels leading to angiogenesis or vascular regression depending on the molecular context in which they act.

The adrenal cortex is a dynamic endocrine organ that exhibits remarkable plasticity depending upon the extent of tropic hormone exposure. Excessive production of corticotrophin (ACTH) by the pituitary (e.g. Cushing’s syndrome) promotes adrenal cortical hypertrophy, whereas the lack of ACTH results in cortical atrophy (13, 14). As with other endocrine organs, the adrenal cortex requires an intense vasculature to facilitate access of hormone products to the circulation (15). Consequently, glucocorticoids synthesized by the steroidogenic cells after ACTH stimulation can easily move from the cells into the intravascular compartment. During experimental and pathological, changes in adrenal cortex size caused by ACTH overproduction or deficiency, the vasculature must evolve in a coordinated manner with trophic regulation of the cortex, so that blood vessel formation and cortical growth are synchronized. Several experimental approaches over the last decades have suggested the potential role of ACTH in maintenance of adrenal vasculature. Indeed, several factors responsible for regulating the development of the vasculature are expressed in adrenal and up-regulated by ACTH (16, 17, 18, 19). Among them, we have shown the expression of the two major transcripts encoding the 121 and the 165 amino acid-long isoforms of VEGF in adrenocortical cells in primary cultures in vitro. ACTH rapidly, within 2–4 h, up-regulated (2- to 3-fold) both VEGF isoforms (17). Furthermore, the expression of the signaling VEGF receptors R1 (flt-1) and R2 (Flk-1) was observed in the adrenal cortex and found to be restricted to endothelial cells (19). We demonstrated more recently the expression of VEGF receptors in mouse adrenal in vivo. We showed that Flk1 was up-regulated in vivo in mouse adrenal, whereas Flt1 was not (20). Another group has recently identified an angiogenic mitogen selective for endocrine gland endothelium (21). Although this protein does not show any structural homology to the VEGF family, it displays several striking biological similarities to VEGF, including hypoxic regulation and the ability to induce fenestration in the target cells (21). Its potential hormonal regulation was not documented. Thus, as ACTH is the major regulator of adrenal gland, and because no angiogenic properties of ACTH per se were demonstrated so far, it is likely that adrenal cortical vasculature is under the control of specific factors. The Tie2/Ang pathway that plays a critical role in angiogenesis in the adult, might be a candidate as a regulatory system of the adrenal vasculature. We examined this possibility by seeking for the expression of Tie2 receptor, and its ligand Ang-1 and its endogenous antagonist Ang-2, and for their response when angiogenesis and growth of the adrenal are pharmacologically altered. Findings described in the current study are the first to our knowledge to demonstrate that Ang-1, Ang-2, and Tie2 are expressed in adult adrenal. We further show that Tie2 receptor is expressed in endothelial cells of adrenal gland. Moreover, we found that DEX treatment, known to impair hypothalamo-pituitary axis in vivo, decreased Ang-1 and Tie2 expression, but did not change that of Ang-2. ACTH administration was able to restore Ang-1 and Tie2 expression, suggesting that the adrenal Tie2/Ang system could be an effective mechanism for coordinating angiogenesis and cortical growth.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
ACTH (1–39), DEX, Triton X-100, and Tween 20 were purchased from Sigma (St. Louis, MO). Collagenase B and deoxyribonuclease I were obtained from Roche Diagnostics (Ottweiler, Germany). The RNAgents Total RNA Isolation System was purchased from Promega (Madison, WI). Hybond N+ and the enhanced chemiluminescence detection reagents were purchased from Amersham (Les Ulis, France). Nitrocellulose was obtained from Schleicher and Schuell (Ecquevilly, France). Micro BCA protein assay reagent kit was purchased from Pierce (Oud Beijerland, The Netherlands). Platelet endothelial cell adhesion molecule (PECAM) antibody (MEC-13.3 hybridoma supernatant) was a generous gift from Dr. A. Vecchi (Mario Negri Istituto, Milan, Italy), the mouse monoclonal antibody against murine Tie2 receptor was purchased from BD PharMingen (San Diego, CA), the fluorescein isothiocyanate-conjugated F(ab')2 fragment of goat antimouse IgG and cyanine 3-conjugated F(ab')2 fragment of goat antirat IgG were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA). Fluorsave was obtained from Calbiochem (San Diego, CA).

Animals
In vivo studies.
Two-month-old Swiss female mice (Charles River Laboratory, Les Oncins, France) (20–25 g) were housed at constant temperature and in an automatically controlled 12-h light cycle environment. Animals were fed chow and water ad libitum and allowed to acclimatize to a 12-h light cycle for a period of 1 wk before experimental manipulation. All protocols in the studies described here were conducted in strict accordance with the Ministère de l’Education Nationale, de la Recherche et de la Technologie Guidelines for the Care and Use of Laboratory Animals.

DEX and ACTH treatment.
Animals were divided in three groups (n = 5 per group) with equal average body weight between the three groups. The first group [control (CTL)] was treated daily by ip injection (200 µl) of sterile 0.9% saline solution for a consecutive 6 d. Animals of the second group (DEX), were treated daily by ip injection of DEX (0.5 mg/100 g body weight) in 0.9% saline for a consecutive 6 d. Animals of the third group (DEX-ACTH), received a daily ip injection of DEX as the second group and were then treated daily by ip injection of ACTH (1–39) (30 IU/kg) for a consecutive 7 d. After the treatments, animals were weighed and then killed by ip application of an overdose of pentobarbital.

Plasma corticosterone measurement.
Fifty microliters of blood withdrawn by cut tail and collected in EDTA-treated tubes. Plasma corticosterone was determined by RIA using an anticorticosterone antibody as described in (22).

Immunofluorescence studies
Adrenal glands were dissected, cut into small pieces and digested by 1 h treatment at 37 C with 0.2% collagenase B, 200 U/ml deoxyribonuclease I, and 10% fetal calf serum in PBS. After two washes in PBS, cells were resuspended in PBS containing 2% BSA at 105 cells/ml. Cytospin preparations of 0.2 ml aliquots of cell suspensions were prepared using a cytospin 2 centrifuge (Shandon S.A., Pittsburgh, PA). Cell smears were air-dried, fixed in acetone (5 min, 4 C), and double stained with anti-PECAM and anti-Tie2 antibodies. After three washes in PBS, slides were incubated with fluorescein isothiocyanate-goat antimouse IgG (1:100) and Cy-3 goat antirat IgG (1:500). Slides were rinsed, counterstained with Hoeschst 33258, to visualize the nuclei, and mounted with Fluorsave mounting media.

RNA preparation and RT-PCR
Adrenal glands were rapidly dissected and stored in ice. Tissues were used immediately for RNA and protein extraction. Total adrenal RNA was isolated using the RNAgents Total RNA Isolation System (23). Semiquantitative RT-PCR was performed as previously described (24). Briefly, first-strand cDNAs (the equivalent of 200 ng reverse-transcribed RNA) were amplified in a final volume of 20 µl with 0.5 U Taq DNA polymerase and 20 pmol of each random hexamers. The amplification parameters were 94 C for 1 min, 59 C for 1 min 72 C for 1 min during 25 cycles for hypoxanthine phosphoribosyltransferase (hprt), followed by 5 min at 72 C for final extension. For the other transcripts, hybridization temperature and number of cycles were, respectively, 55 C and 30 cycles for Tie2, 59 C and 30 cycles for Ang1, and 59 C and 32 cycles for Ang2. All PCR experiments included reverse transcriptase negative controls and a blank with no template. To ensure semiquantitative results, the number of PCR cycles for each set of primers was selected to be in the linear range of amplification. In addition, all cDNA samples were adjusted to yield equivalent amplification of hprt as a reference standard. Hybridized filters were visualized and signals quantified using a Fluorimager (Molecular Dynamics, Sunnyvale, CA). Primers and probes used in these studies are listed in Table 1Go.


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  		TABLE 1. Sequences of oligonucleotide primers and probes used for RT-PCR analyses

 
SDS-PAGE and Western blotting
Proteins isolated from phenol-ethanol supernatants obtained after precipitation of DNA with ethanol (Gibco Invitrogen Life Technologies, Paisley, UK), were analyzed by SDS-PAGE and Western blotting as described in Ref. 25 . Briefly, 80 µg of proteins extract were analyzed by a 10% acrylamide-0.2% bis-acrylamide-sodium dodecyl sulfate gel electrophoresis. Proteins were then transferred from the gel onto nitrocellulose for 1 h and the residual binding sites were blocked with 5% nonfat dry milk and subsequently incubated overnight at 4 C with primary rat monoclonal anti-Tie2 receptor antibody (2 µg/ml in PBS/5% milk/0.05% Tween 20). After washing, specific proteins were then detected using horseradish peroxidase-conjugated rabbit antirat secondary antibody and enhanced chemiluminescence reagents provided with an electrochemiluminescence kit.

Data presentation and statistical analyses
Within each group of animals, three independent measurements of mRNA and protein levels were made for Ang, Tie2 receptors, and hprt. OD of RT-PCR products were normalized (for hprt) and expressed in relative units. The majority of data are presented as the mean ± SEM, and the numbers of subjects in each experimental group are indicated on either the figures themselves or in the figure legends. Significant changes in hormone concentration; adrenal weight and mRNA levels were determined by statistical analyses of these data were performed using either paired or unpaired t test or a one-way ANOVA (followed by Tukey-Kramer multiple comparisons test) as appropriate. In all analyses a two-tailed probability of less than 5% (i.e. P < 0.05) was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of Ang and Tie2 in mouse adrenal tissue
In an effort to elucidate the role of Ang in the regulation of the adrenal vascular bed, we first examined whether their mRNAs were expressed in adult adrenal tissue using RT-PCR analysis. cDNA templates synthesized from RNA preparations from mouse adrenal were amplified using the oligonucleotide primers described in Table 1Go. For the negative control, RNA was substituted by water (no template). As shown in Fig. 1AGo, amplification of Ang-1, Ang-2 transcripts with RT-PCR in adrenal produced a single product ({approx}380 bp and {approx}310 bp long, respectively). These two bands correspond in size to the RT-PCR product expected from mouse Ang-1 mRNA (387 bp), and mouse Ang-2 mRNA (311 bp), respectively.



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  		FIG. 1. Expression of Ang and Tie2 receptor in adult mouse adrenal in vivo. RT-PCR analysis. Total RNA was reverse transcribed and amplified using exponential nonsaturating conditions with specific oligonucleotides for Ang-1 and Ang-2 (A) and for Tie2 (B). PCR products were separated on ethidium bromide-stained 2.5% agarose gel. Arrowheads point to the band corresponding to the Ang isoforms. No template was used as the negative control. The identity of the PCR products for the Ang, Tie2 has been be confirmed and hybridization with their respective [32P]-5'-end-labeled internal oligonucleotide probe. C, Adrenal protein was extracted and 80 µg were analyzed by SDS-PAGE (12% acrylamide) as described in Materials and Methods. Immunoblot analysis was performed with the anti-Tie2 antibody. Molecular mass standards (in Da) are shown at the left. D, Nuclei labeling with Hoechst dye (a), immunolocalization of Tie2 (b), and PECAM (c). These results are representative of a typical experiment out of three experiments performed.

 
As Ang actions are mediated through Tie2 receptor activation, we then examined whether Tie2 was expressed in adrenal at the mRNA and protein level. We performed RT-PCR analysis using oligonucleotide primers designed to amplify mouse Tie2 mRNA (see Table 1Go). The specificity of the amplified product was compared with mouse hemangioma vascular endothelial cells (H5V), which constitutively express Tie2 receptor gene and confirmed by Southern hybridization with a specific internal oligonucleotide probe (24) (data not shown). As shown in Fig. 1BGo, a band of approximately 440 bp corresponding in size to the RT-PCR product expected from mouse Tie2 receptor mRNAs (441 bp), was detected in the adrenal extracts. The presence of the Tie2 receptor protein, was analyzed by Western blotting of mice adrenal protein extracts using an antibody to the entire extracellular domain of Tie2 receptor. As shown in Fig. 1CGo, the antibody recognized a single band with a molecular mass of 140 kDa corresponding to a full-length molecule of Tie2 protein. To confirm and to expand the data obtained by RT-PCR and Western blot analysis, we examined whether adrenal endothelial cells expressed Tie2 receptor. To this end, cytospin preparations of cell suspensions from adrenal tissue were double immunostained with an antibody against the platelet endothelial cell adhesion molecule PECAM (CD31), a marker of endothelial cells (26) and an antibody against murine Tie2. As shown in Fig. 1DGo, immunoreactive Tie2 and PECAM show a similar cellular pattern of staining in the adult adrenal endothelial cells. These data were confirmed by analysis of PECAM and Tie2 immunolocalization in whole adrenal gland where the colocalization of the two endothelial markers was also observed (data not shown). Taken together, these results indicate that Tie2 receptor and its ligands Ang-1 and Ang-2 are expressed in the adult mouse adrenal in vivo, suggesting a functional role of Ang in the adult endocrine tissue.

Role of the pituitary adrenocortical axis on the expression of the Tie2/Ang system
Several experimental approaches over the last few decades have clearly established that the pituitary hormone ACTH, is the primary regulator of both fetal adrenal development and adult adrenal cortex homeostasis. To assess the mechanisms that might regulate Ang-Tie2 expression in mouse adrenal in vivo, DEX, a synthetic glucocorticoid that suppresses the pituitary adrenocortical axis, was used to induce ACTH down-regulation and adrenal regression in rat in vivo (27, 28). Animals received daily injections of DEX (0.5 mg/100 g body weight ip) for a consecutive 6 d (DEX group) or saline (CTL group). As an internal control of the effect of DEX in mice, the adrenals of each animal in the two groups were weighed. DEX induced a strong decrease in adrenal weight from 9.77 ± 0.78 mg in CTL group to 4.8 ± 0.60 mg in the DEX group. Using RT-PCR analysis, we found a lower level in ACTH receptor transcripts in the DEX group (data not shown). This was confirmed at the protein level because the presence of the 36-kDa protein specific for MC2 receptor was detected in adrenal extracts from CTL group, whereas it was barely detectable in the DEX group (Fig. 2AGo). As shown in Fig. 2BGo, the lower level of ACTH receptor expression was associated with a significantly (P < 0.001) lower plasma corticosterone level in DEX group (114 ± 18 ng/ml) as compared with the CTL group (444 ± 57 ng/ml). Thus, these results show that DEX treatment induced pharmacological modifications of the mouse adrenal. Previous reports in rats have shown that the dramatic regression of the adrenocortical tissue was caused by a massive deletion of the fasciculata and reticularis zones through apoptosis, whereas the glomerulosa zone remains intact and functional (13, 29, 30, 31).



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  		FIG. 2. Effects of DEX administration on adrenal ACTH receptor expression and plasma corticosterone level. Groups of female mice (n = 5) were injected ip with 200 µl saline or DEX (0.5 mg/100 g body weight) daily for 6 d, before collection of blood at the tail. Adrenal of each animal were dissected. A, Adrenal protein was extracted and 80 µg were analyzed by SDS-PAGE (12% acrylamide) as described in Materials and Methods. Immunoblot analysis was performed with the anti-MC2 receptor antibody. Molecular mass standards (in Da) are shown at the left. B, The individual concentrations of plasma corticosterone were determined and were significantly reduced in DEX treated mice (P < 0.001).

 
The level of Ang-1 and Ang-2 transcripts was then analyzed in this model of adrenal regression induced by DEX in vivo. Figure 3Go, A and B, illustrates the changes in Ang expression in the adrenal of CTL group and DEX group. RT-PCR analyzes show a significant decline in Ang-1 mRNA transcripts in DEX group compared with normal control (CTL), whereas Ang-2 mRNA remained constitutively expressed (Fig. 3BGo). Figure 3CGo illustrates analysis of mean ODs of Ang-1, -2 mRNA levels relative to OD of hprt. In CTL group, the Ang-1/hprt relative ratio was 1.65 ± 0.12 and Ang-2/hprt was 1.5 ± 0.1. In DEX group, the value for the ratio Ang1/hprt was 0.42 ± 0.05, whereas the value for the ratio Ang2/hprt was 1.47 ± 0.15. As the relative ratio of levels of Ang-2 vs. Ang-1 has been suggested to be critical in the functional regulation of Ang (11, 31), we thus calculated the relative Ang-2 /Ang-1 ratio in the two groups. As shown in Fig. 3DGo, in the CTL group, the Ang-2/Ang-1 relative ratio was 0.90 ± 0.05, whereas in the DEX group, it was 3.53 ± 0.15). These in vivo observations support the view that DEX-induced-ACTH down-regulation significantly (P < 0.05) modulates the Ang2/Ang1 relative ratio in mouse adrenal.



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  		FIG. 3. Regulation of Ang-1, Ang-2 mRNA expression during normal and DEX-induced adrenal atrophy. Expression of Ang, Ang-1 (A), and Ang-2 (B), is shown for CTL mice and DEX-treated mice, as indicated. Total RNA was reverse transcribed and amplified using exponential nonsaturating conditions with specific oligonucleotides for Ang-1 and Ang-2. PCR products were separated on ethidium bromide-stained 2.5% agarose gel. Arrowheads point to the band corresponding to the isoforms for Ang. No template was used as the negative control. Expression of hprt mRNA is shown as a loading control. C, Analysis of mean ODs of Ang-1, Ang-2 mRNA levels relative to optical density of hprt in adrenal glands of CTL and DEX group expressed in relative units. D, Relative Ang-2 to Ang-1 ratio. Groups denoted by the same letter above the column were not statistically significantly different (by one-way ANOVA, followed by Tukey-Kramer multiple comparisons test).

 
Reversal of DEX effect by ACTH
To determine whether ACTH was involved in the observed effects of DEX on adrenal expression of Ang, we examined the reversibility of DEX treatment by daily injection of ACTH1–39 for a consecutive 7 d to DEX-treated mice (DEX-ACTH). The physiological response of adrenal gland to ACTH injections was monitored by RIA of plasma corticosterone every day for 7 d. As shown in Fig. 4Go, the administration of ACTH to the DEX group (DEX-ACTH) led to an increase in plasma corticosterone in a time-dependent manner. The effect was already detected by d 1 (142 ± 12 ng/ml), then significantly increased by d 3 (235 ± 25 ng/ml) (P < 0.01), to reach by d 7, the levels of corticosterone measured in the CTL group (465 ± 35 ng/ml) (P < 0.001). Injection of saline in the CTL group did not lead to a detectable increase in blood hormone levels. Furthermore, the absolute weights of the adrenal were significantly greater in the DEX-ACTH group after 7 d of ACTH injections (8.2 ± 0.5 mg) (n = 10) then in the DEX group (data not shown). These physiological parameters strongly suggest that 7 d of ACTH treatment to DEX-treated mice has completely restored the steroidogenic function of the adrenal. RT-PCR analysis of Ang transcripts in adrenal of the DEX-ACTH group and densitometric analysis of relative Ang vs. hprt levels are illustrated in Fig. 5Go. In the DEX-ACTH group, the Ang1/hprt relative ratio was 1.14 ± 0.07, whereas the value for the ratio Ang2/hprt was 1.65 ± 0.16 (Fig. 5Go, A–C), which shows that ACTH treatment resulted in a significant (P < 0.05) increase in Ang-1 mRNA accumulation. As shown in Fig. 5Go, B and C, no significant variations in Ang-2 mRNA expression was observed (DEX-ACTH Ang2/hprt: 1.68 ± 0.18). The relative Ang-2/Ang-1 ratio in DEX-ACTH group was 1.44 ± 0.16 (Fig. 5DGo). This result indicates that ACTH treatment restored Ang-2/Ang-1 value near to CTL group. Altogether, these observations strongly suggest that ACTH plays a key role in Ang-1 expression in adrenal gland in vivo.



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  		FIG. 4. Plasma corticosterone responses to ACTH in female mice treated with DEX. The CTL group of mice (n = 5) was injected ip with 200 µl saline every day for 6 d and the DEX-ACTH group of mice (n = 5) was injected ip with 200 µl ACTH(1 IU/d). The blood was collected at the tail every day. Corticosterone was determined for each mouse in triplicate, as described in Materials and Methods section. Kinetics of plasma corticosterone concentration are represented.

 


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  		FIG. 5. Effect of ACTH administration on adrenal Ang mRNAs in mice pretreated with DEX for 6 d. A and B, Total RNA from whole adrenal of the DEX and DEX-ACTH groups of mice was reverse transcribed and amplified using exponential nonsaturating conditions with specific oligonucleotides for Ang-1 and Ang-2. Arrowheads point to bands corresponding to each Ang isoform. One representative experiment is shown; expression of hprt mRNA is shown as a loading control. C, Analysis of mean ODs of Ang-1, Ang-2 mRNA levels relative to OD of hprt expressed in relative units in DEX-treated mice injected (DEX-ACTH) or not (DEX) with ACTH. D, Analysis of Ang-2/ Ang-1, ratio in adrenal glands of normal and DEX-treated mice expressed in relative units. Groups denoted by the same letter above the column were not statistically significantly different (by one-way ANOVA, followed by Tukey-Kramer multiple comparisons test).

 
Hormonal regulation of Tie2 expression
Because the activities of Ang are exerted through their specific Tie2 receptor, we investigated next whether the Tie2 receptor mRNA and protein expression was under ACTH control in vivo. Figure 6AGo illustrates the changes in Tie2 receptor mRNA expression in adrenal from CTL, DEX, and DEX-ACTH groups. Analysis of the mean ODs of Tie2 mRNA levels expressed in relative units (relative to OD of hprt) shows that DEX treatment resulted in a strong decrease of Tie2 mRNA expression (by 60%, P < 0.01), compared with the CTL group. ACTH therapy to DEX-treated mice induced a significant rise (P < 0.05) in Tie2 mRNA expression to reach the level measured in CTL group. To ascertain the regulation of Tie2 receptor at the protein level, immunoblotting was performed on 80 µg of adrenal protein extracts from CTL, DEX, and DEX-ACTH groups. As shown in Fig. 6CGo, DEX treatment induced a strong decrease in the 140-kDa Tie2 protein. Although a proteolytically degraded form of Tie2 could be recognized by the anti-Tie2 antibody, no degradation products that could account for the decreased level of Tie2 protein in DEX group were seen (data not shown). The Tie2 protein level detected in adrenal from the DEX-ACTH group was similar to the level detected in the CTL group.



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  		FIG. 6. Effects of DEX and ACTH on Tie2 receptor expression. A, RT-PCR analysis. Total RNA from whole adrenal of the three groups of treated mice (CTL, untreated; DEX; DEX-ACTH, DEX followed by ACTH) was reverse transcribed and amplified using exponential nonsaturating conditions with specific oligonucleotides for Tie2. Arrowheads point to bands corresponding to the Tie2 mRNA receptor product. B, Tie2 mRNA were quantitated by Fluorimager scanning and normalized to the hprt signals Groups denoted by the same letter above the column were not statistically significantly different (by one-way ANOVA, followed by Tukey-Kramer multiple comparisons test). C, Regulation of Tie2 protein expression in normal and treated animals. Total protein (80 µg) from adrenal lysates of each group were assessed for the amount of Tie2-specific proteins by western blotting. Four adrenals (n = 4) were excised for each experimental time point and used for protein isolation. Arrows show the mature form of Tie2 (140 kDa).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The expansion or remodeling of preexisting blood vessels, known as angiogenesis, by either nascent sprouting, intercalated or intussusceptive growth is a highly regulated process. Angiogenesis is critical not only during normal embryonic vascular development, but also in the progression of several diseases, including cancer, psoriasis, and diabetes. Mouse molecular genetic experiments have shown that the Ang and their receptor Tie2/Tek are indispensable for embryonic vessel development (8, 9). The importance of the Ang-signaling pathway has also been shown to extend beyond development, in experimental models of angiogenic growth both in vivo and in vitro (3, 4). Currently the precise role of the Ang remains unclear. However, what is emerging from genetics, xenograft transplant, histochemical, and cell culture experiments is that the response of endothelial cells to Ang appears to be context and endothelial cell type specific (10, 11, 32).

The physiological characteristics of the adrenal vasculature and the tight endocrine regulation of the gland by adrenocorticotropic hormone offers a unique experimental system to analyze the hormonal control of blood vessel growth, maturation, and regression. Increasing evidence have suggested that ACTH, which is not an angiogenic factor per se, regulates the adrenal vasculature through its action on growth factors synthesis (16, 17). Although many of the genetic manipulations in mice demonstrate a critical role for the Ang-R pathways in vasculogenesis, it is impossible to investigate their role in adrenal function in these models in that the embryonic defects are lethal. Nevertheless, we have investigated whether the components of these endothelial cell regulatory pathways are expressed in the adrenal, and if so, what regulates their expression.

We demonstrate for the first time that Tie2 is expressed in the adrenal endothelial cells in adult mouse that is consistent with its largely endothelial-restricted expression shown by studies in endothelial, hematopoietic, and leukemia blood cells (33) in vitro and in quiescent adult tissues in vivo (8, 34). Previous studies in adult rat have shown that Tie2 was homogeneously expressed throughout the vasculature in the endothelium of arteries, veins, and capillaries of ovary, kidney, and skeletal muscle (34). Similarly, microvascular endothelial cells in the brain, heart, and spleen demonstrated prominent expression of Tie2. Maintenance functions have been suggested for other endothelial receptor tyrosine kinases. For example, Flt-1, a VEGF receptor, is also broadly expressed in the adult vasculature (35). The results of the present study demonstrating Tie2 protein expression in mouse adrenal suggests a role for Tie2 signaling in the maintenance of the quiescent adult vasculature. The endothelial restriction of Tie2 is thought to be important for the biological actions of the Ang. Conceivably, during adrenal development, the growth, differentiation, and reorganization required of the adrenal cells may compare with those in embryogenesis (8). In contrast, the exact role played by this pathway in the mature adult vasculature remains unclear.

Previous studies have shown that Ang-1 is widely expressed in mouse, and Ang-2 appears to be primarily expressed in the ovary, uterus, and placenta (3). The adrenal gland was not investigated yet, we demonstrate in this work that Ang-1 and Ang-2 are both expressed in the adult adrenal tissue. Histological studies have shown that Ang-1 protein is present on the endothelium of blood vessels that supports the paracrine mode of action of this ligand-receptor system (36). Due to the lack of suitable antibodies, the identification of cell type-specific Ang-1, Ang-2 expression in adrenal tissue sections was unsuccessful in our hands. Moreover, the close proximity of endothelial cells and pericytes has often made it difficult to determine with certainty the specific cell type(s) that expressed each of these proteins in situ (37). Ang-1 was recently described to be expressed by pericytes in vitro and in vivo (37). In contrast, although vascular expression of Ang-2 has been described previously, the exact cell type has not been defined. From previous experiments in E12.5 mice (4), it is known that Ang-2 transcripts are associated with vascular structures. In adrenal, detailed studies on protein production and receptor activity are needed, as well as definition of the cell type(s) producing Ang and the target cells with their receptors. However, this early report indicates that ligand-receptor systems exist for Ang-1/-2 within the adrenal of adults mice. Further investigations are needed to determine whether Ang-1 acting through Tie2 increases the growth of endothelial cells, or prolongs cell survival (36, 38), or coordinates steroidogenic and endothelial cell development, and/or regulates steroidogenic cell function.

In our mouse model of steroid-induced adrenal regression, we observed a dynamic expression of Ang-1 and Ang-2 in vivo. Indeed, we found a decreased expression of Ang-1 and Tie-2 receptor mRNA, whereas Ang-2 mRNA remained constant. Because the neovascularization process is dependent on the dominance of angiogenic mediators over inhibitors, our findings of a modified Ang-2/Ang-1 mRNA ratio in the adrenal of DEX-treated mice is of importance. Indeed, it was shown in several experimental models that exogenous administration of Ang-2 induced endothelial cell death and vessel regression. Moreover, the decreased level of Tie2 receptor expression along with the reduction in the Ang-1 would be expected to reduce Tie2 signaling. In this context, in adrenal of DEX-treated mice, our observations support a model in which the presence or absence of Tie2/Ang-1 alters the action of Ang-2 resulting in a vascular instability. The underlying causal mechanisms of the changes that we observed remain to be determined. Previous studies have shown that long-term DEX treatment negatively regulate CRH gene expression (39) and hence ACTH circulating level. Thus, our results suggest that in vivo variations of ACTH secretion, could regulate the expression of endothelial specific effectors. This was confirmed in particular by our finding showing that exogenous administration of ACTH leads to up-regulation of Ang-1 expression. Similar results were described in primate granulosa cells where increased levels in Ang-1 mRNA were found after the onset of the ovulatory gonadotropin stimulus, whereas Ang-2 levels remained unchanged (40). Thus, our results in adult adrenal tissue would suggest that ACTH exerts a permanent positive effect on Ang-1 expression to trigger a stabilizing effect through Tie-2 receptor in mature adrenocortical endothelial cells.

Our in vivo experiments demonstrate that Tie2 receptors are under hormonal control in adult adrenal. Other studies have shown the presence and up-regulation of Tie2 mRNA in several pathological situations such as metastatic melanomas (41), during ovulation and wound healing (42), in breast cancer vasculature (43), and in reperfusion after cerebral ischemia (44). However, no clear mechanism has been identified for these increases. The mechanisms through which ACTH modulates Tie2 and Ang-1 expression in this study remain also to be determine. In the rat thyroid, the up-regulation of Tie2 mRNA by TSH has been reported (45). This effect of TSH was found to be mediated by the cAMP pathway. In the adrenal, the effects of ACTH are transduced through elevations in cAMP, thus cAMP could be thought of as a regulator of Tie2 expression in the adrenal. However, it should be made clear whether such control by ACTH is direct in endothelial cells or mediated by another relay from steroidogenic to endothelial cells (46). Taken together, our data suggest a role for Tie2 signaling in the maintenance of the quiescent adult vasculature.

In conclusion, our descriptive data are consistent with the hypothesis that Ang-1 may have multiple roles in the maturation of the adrenal microcirculations. We speculate first that, as an adjunct to a strong mitogenic effect of VEGF on endothelial cells, the chemotactic effect of Ang-1 (47) may be involved in recruiting endothelial cells to initiate and accelerate reendothelialization during regeneration of the adrenal. Second, the expression of Ang-1 may by important for the maturation of cortical capillaries that express Tie2. Although our results lead to speculation regarding the roles of the Ang and Tie2 in the adrenal vasculature, definitive proof of their roles awaits functional data. Unfortunately, the relatively early embryonic death of Ang-1 and Tie2 null mutant mice precludes analyses of the potential roles of these genes during adrenogenesis. In the future, an analysis of the adrenals of chimeric mice constituted from null and wild-type cells may be useful. In addition, further work is clearly needed to fully document the potential expression of the Ang and Tie2 in the adrenal development. Furthermore, we still know very little concerning factors critical for vascular changes during adrenal regression or hyperfunction. This is essential from a clinical standpoint because it is likely (based on studies in other vascular beds) that either under- or overproduction of angiogenic factors will cause microvascular dysfunction and will play a role in adrenal disorders. Further information on the regulation and roles of angiogenic factors and their receptors could yield novel strategies for manipulating the adrenal vasculature. It is clear, however, that the demonstration of Ang-1 gene expression in the mouse adrenal and its related down-regulation in DEX-induced adrenal atrophy establishes this family of angiogenic growth factors as important regulators of adrenal function.


   Acknowledgments
 
We gratefully acknowledge Drs. Daniel Vittet and Georges Christé for the critical reading and comments on the manuscript. We are grateful to Dr. Geneviève Defaye for providing us with the anticorticosterone antibody.


   Footnotes
 
This work was supported by the INSERM (Unité 244, EMI 01-05, EMI 02-19), the Commissariat à l’Energie Atomique, Direction des Sciences du Vivant/Département Réponse Dynamique Cellulaire, by the Association pour la Recherche contre le Cancer (ARC No. 5588), the Fédération Nationale des Centres de Lutte contre le Cancer, the Fondation pour la Recherche Médicale, and the Ligue Nationale contre le Cancer.

Abbreviations: Ang, Angiopoietin; CTL, control; EC-VEGF, endocrine-gland-derived vascular endothelial growth factor; EGTA, ethylene bis (oxyethylene nitrilo) tetraacetic acid; Flk, fms-like tyrosine kinase; Flt, fetal liver tyrosine kinase; hprt, hypoxanthine phosphoribosyltransferase; PECAM, platelet endothelial cell adhesion molecule; Tris, tris (hydroxymethyl) aminomethane; SDS, sodium dodecyl sulfate; Tie2, tyrosine kinase with Ig and epidermal growth factor homology; VEGF, vascular endothelial growth factor.

Received January 21, 2003.

Accepted for publication June 10, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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