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research-article |
Department of Obstetrics and Gynecology, University of Texas Medical Branch, Galveston, Texas 77555
ABSTRACT
Adrenomedullin 2 (ADM2) is a recently discovered member of the calcitonin/calcitonin gene-related peptide family with an exon-intron structure similar to that of ADM. The mRNA of ADM2 is expressed in several tissues, including uterus and ovary. The present study was designed to assess the effects of ADM2 antagonist (ADM217–47) infusion to pregnant rats on fetal and placental growth. On Day 15 of gestation, rats were implanted s.c. with osmotic minipumps delivering 50 and 200 µg per rat per day of ADM217–47 and were killed on Gestational Day 18. In ADM217–47-treated rats, placental weights were significantly inhibited in a dose-related manner, with an 11% reduction in the group of rats receiving 200 µg/day, whereas the fetal weights were reduced by 17% without significant differences between the two doses. 2 In ADM217–47-infused rats, increased apoptosis was demonstrated in the labyrinth and junctional zones of rat placenta by the TUNEL method compared with the control animals. Western blot analysis demonstrated that in ADM217–47-treated rats Bcl-2, mitochondrial cytochrome c, and active caspase-9 and caspase-3 were significantly increased compared with the controls. No significant treatment-associated changes were observed in Bax, Bid, p53, and caspase-8 and caspase-10 proteins in the treated placentas. In addition, infusion of ADM217–47 caused a significant decline in the transcripts of nitric oxide synthase 3 (NOS3) and NOS2. These findings show that ADM217–47 infusion in rats during midpregnancy cause fetoplacental growth restriction through the activation of mitochondrial apoptotic pathways. This study demonstrates for the first time (to our knowledge) a potential role for ADM2 in placental functions during pregnancy.
ADM, ADM2, apoptosis, fetus, placenta, pregnancy
Calcitonin, calcitonin gene-related peptide (CALCA), adrenomedullin (ADM), and amylin belong to a unique group of calcitonin/CALCA family peptide hormones that are important for homeostasis in diverse tissues. ADM2 is a 47-amino acid novel calcitonin/CALCA family peptide discovered simultaneously and independently by two groups of investigators from the genome of humans and other vertebrates in 2004 [1, 2]. Based on phylogenetic analysis, ADM2 and ADM fall into two distinct but closely related groups with 33% structural homology. Pharmacological analyses show that ADM2 binds to and activates CALCA and ADM receptor complexes comprising a 7-Transmembrane domain class B G protein-coupled receptor (GPCR), calcitonin receptor-like receptor (CALCRL), and receptor activity-modifying proteins (RAMPs) [1]. The combination of CALCRL and RAMP1 gives rise to CALCA receptor, whereas CALCRL in combination with RAMP2 or RAMP3 forms ADM receptors [3–6]. ADM2 is a nonselective agonist for RAMPs, as CALCRL in combination with any one of the three RAMPs forms ADM2 receptor but exhibits greater potency with CALCRL/RAMP1 and CALCRL/RAMP3 [1].
Recent investigations show that the ADM2 promoter sequence contains consensus estrogen response elements and that ADM2 is an estrogen-dependent prolactin-releasing factor [7]. Peripherally administered ADM2 exhibits potent hypotensive effects and has suppressive effects on gastric emptying activities and on food and water intake [1]. In addition, ADM2 stimulates vasodilatory activity in pulmonary vascular beds of rats under elevated vascular tension [8]. Therefore, ADM2 is a novel ligand that is specific for CALCRL/RAMP1 and CALCRL/RAMP3 receptors and could be important for regulation of diverse physiological processes that have been hitherto attributed to CALCA and ADM [1, 3, 7, 9–11]. Although all three ligands, CALCA, ADM, and ADM2, are capable of interacting with CALCRL, optimal regulation by the GPCR signaling pathway likely depends on an integrated release of different endocrine/paracrine ligands in a tissue-specific and time-coordinated manner. Because ADM2 can activate ADM and CALCA receptors, some of the ADM2 actions may be more potent than those of CALCA or ADM. Concentrations of ADM2 in rat and mice plasma are higher than those of ADM [11]. Northern blot and immunohistochemical analysis have shown that ADM2 is expressed in anterior lobes of the pituitary [1], suggesting that ADM2 could function as an endocrine factor or a paracrine factor regulating pituitary hormone secretion. ADM2 is reported to be present in several other tissues such as the hypothalamus, lung, kidney, brain, heart, stomach, thymus, ovary, and uterus [1, 2, 12]. Demonstration of ADM2 as an estrogen-dependent regulator of prolactin, together with expression of ADM2 in the uterus and ovary [2, 7], suggests a possible role for this novel peptide in female reproductive functions.
Moriyama et al. [13] reported that the expression of ADM, an ADM2-related peptide, in the cytotrophoblast is most abundant in the first-trimester human placenta. It was recently reported that infusion of ADM antagonist to rats from Day 14 of pregnancy caused detrimental effects on fetal and placental growth, impaired the placental vasculogenesis, and stimulated an increase in apoptotic markers in the placenta [14, 15]. Therefore, we hypothesized that ADM2 is required for normal fetoplacental growth and that the suppression of ADM2 actions would be detrimental to fetoplacental growth. In this study, we followed a strategy similar to that of a previous study performed with ADM [15]. ADM2 antagonist, ADM217–47, was continuously infused from Day 15 of gestation, and fetal and placental growth was assessed on Day 18. Because many biological functions of calcitonin/CALCA family peptides are mediated through nitric oxide (NO) and because ADM2 activates the L-arginine/NO pathway in rat aortas and dilates rat pulmonary vasculature via NO release [8, 16–18], we assessed the effect of ADM217–47 infusion on transcript levels of placental NO synthase 3 (NOS3) and NOS2. In addition, we examined the effect of ADM217–47 on the expression profile of proapoptotic and antiapoptotic proteins involved in intrinsic and extrinsic apoptotic pathways in rat placenta. Because DNA damage in apoptosis could lead to an increase in tumor suppressor protein TRP53, we analyzed whether ADM217–47 has any effect on the expression of TRP53 protein in rat placenta.
Female timed pregnant rats (body weight, 200–300 g) were purchased from Harlan Sprague Dawley (Houston, TX). All animals were housed in a climate-controlled room with a 12L:12D schedule and were fed standard rat chow with water to drink ad libitum. All procedures were approved by the Animal Care and Use Committee of the University of Texas Medical Branch (Galveston, TX). Four to five pregnant rats were used in each experimental group.
On Day 15 of gestation, osmotic minipumps (model 2ML1, 10 µl/h; Alza, Palo Alto, CA) were inserted s.c. into the dorsum of pregnant rats while animals were under anesthesia. Anesthesia consisted of a combination of ketamine (45 mg/kg of body weight; Fort Dodge Laboratories, Fort Dodge, IN) and xylazine (5 mg/kg of body weight; Burns Veterinary Supply, New York, NY). The minipumps were filled with saline alone or with saline containing different concentrations of ADM217–47. These concentrations were chosen to deliver ADM217–47 at 50 and 200 µg per rat per day. Based on the pumping rate and the duration of infusion, we prepared the drug concentrations in the pumps to provide the specified daily dose of the drug. All rats were killed on Gestational Day 18 using a CO2 inhalation chamber. The dose and the Day 18 time point selected in this study were based on previous investigations performed with ADM [15]. Placentas and fetuses were carefully dissected out, and weights were recorded. In addition, placentas were collected for hematoxylin-eosin (H&E) staining, immunohistochemistry, and Western blot analysis. Tissues from animals receiving 200 µg/day of ADM217–47 and from vehicle-infused control animals were fixed in Bouin fluid for immunohistochemistry or were frozen in liquid nitrogen and stored at –80°C for further analysis of various proteins.
In Situ Detection of DNA Nicking
Tissue samples were embedded in paraffin, and sections (thickness, 5 µm) were cut with a microtome and placed on coated slides. Paraffin was removed from the tissue sections with xylene, and the sections were rehydrated in graded ethanol solutions. DNA fragmentation was detected by the TUNEL method, using an Apop Tag Kit (Oncor, Gaithersburg, MD) according to the manufacturer's instructions. After the tissue sections were deparaffinized, protein was digested with 20 µg/ml of proteinase K for 15 min at room temperature. Endogenous peroxidase activity was quenched with 3% H2O2 in PBS. After washing with PBS, an equilibration buffer was applied directly to the specimen. Terminal deoxynucleotidyl transferase (Dntt) enzyme and deoxyuridine triphosphate (Dut) digoxigenin were added and incubated at 37°C for 1 h in a humidified chamber. The reaction was then stopped with a stop/wash buffer supplied with the kit, and the slides were incubated with an antidigoxigenin-peroxidase solution for 30 min at room temperature, colorized with diaminobenzidine/H2O2, and counterstained with methyl green. Negative controls were processed with labeled Dut in the absence of Dntt enzyme.
H&E staining. For this portion of the study, placentas were collected from rats infused with 200 µg/day of ADM217–47 or with vehicle control. The placentas were fixed in Bouin solution, and a 2-mm perpendicular section through the central portion of the placenta was processed, embedded in paraffin, and cut into 6-µm sections. Sections were stained with H&E. The maternal decidua and placental labyrinth were evaluated for any ADM217–47-associated morphological changes.
RNA extraction and RT-PCR. To determine whether ADM217–47 infusion alters the expression of NOS3 and NOS2 in placenta, the transcript levels for these NOS enzymes were measured by RT-PCR. Trizol reagent was used to isolate tRNA from ADM217–47-treated and control rat placentas as per the manufacturer's protocol. The RNA was dissolved in 20 µl of ribonuclease (RNase)-free water containing deoxyribonuclease (DNase) I buffer and 2 units of amplification grade DNase I. The DNase I was removed by phenol chloroform extraction. The RNA was dissolved in RNase-free water and was stored at –70°C until use. Using tRNA, first-strand cDNA was synthesized by RT in a 20-µl reaction volume containing PCR buffer, reverse transcriptase, RNase inhibitor, 2 µg of RNA, 5 mM MgCl2, 1 mM deoxyribonucleotide triphosphate (dNTP) mixture, and random primers as described by the supplier (Ambion Inc., Austin, TX). For RT, samples were placed into a thermal cycler for 1 cycle at 28°C for 15 min, 42°C for 45 min, 99°C for 5 min, and 4°C for 5 min. The cDNA was stored at –20°C.
The PCR reactions were initiated for NOS3 and NOS2 by the specific primer set designed based on the published DNA sequence. Briefly, 2 µl of cDNA was mixed with a PCR mixture containing 2.5 mM MgCl2, 1x of 10x PCR buffer, 5 U/100 µl of 1 mM dNTP mixture, and 0.2 µM of the following gene-specific primers: NOS2 forward: 5'-GAATACCAGCCTGATCCATGGAA-3' and reverse: 5'-TCCTCCAGGAGGGTGTCCACCGCATC-3',3); and NOS3 forward: 5'-GGACTTCATCAATCAGTACT-3' and reverse: 5'-GATGTAGGTGAACATTTCC-3'. The PCR cycle involved an initial denaturing step at 95°C for 5 min, followed by 35 cycles at 94°C for 30 sec, 50°C for 30 sec, 73°C for 2 min, and 72°C for 7 min. Amplification of the housekeeping gene 18S was also performed for the same samples using a standard 18S primer pair (Ambion).
The PCR reactions were carried out on a GeneAmp PCR system 9700 (Perkin Elmer, Branchburg, NJ). The PCR products were visualized on a 1.4% agarose gel containing 0.5 µg/ml of ethidium bromide, run in 0.5x Tris-borate-EDTA buffer at 100 V for 1.5 h. Gels were placed on a UV light box and imaged, and expression of NOS2 and NOS3 transcripts were analyzed relative to the 18S with the Sigma gel (SPSS Inc.).
Tissue Preparation and Subcellular Fractionation
Placental tissues (100 mg) were homogenized in 500 µl of Tris buffer (50 mM Tris, 0.1 mM EGTA, 100 mM PMSF, 1.4 µl of ß-mercaptoethanol, and 1 mini-tablet of protease inhibitor cocktail per 10 ml) with polytran at 15 000 rpm for 10 sec. After centrifugation of the homogenate at 1000 x g for 10 min at 4°C, the supernatant fraction was aliquoted and stored at –80°C. For subcellular fractionations and to obtain mitochondria, the supernatant was centrifuged at 10 000 x g for 20 min. The mitochondrial pellet was washed three times in homogenizing buffer and then solubilized in TNC buffer (10 mM Tris acetate, pH 8.0, 0.5% Nonidet p-40, and 5 mM CaCl2) containing protease inhibitors. Protein concentration was determined by bicinchoninic acid kit.
Equal amounts of protein (40 µg) were separated by various appropriate concentrations of SDS-PAGE: 12% for Bcl-2, Bax, Bak1, Bad, Bid (catalog no. SC-529), caspase-8, caspase-9, caspase-10, PARP1, and TRP53 and 15% for cytochrome c and caspase-3. Gels containing the SDS-PAGE-separated proteins were equilibrated in transfer buffer (25 mM Tris, pH 8.3, 190 mM glycine, 0.05% SDS, and 20% methanol) and were electrotransferred to nitrocellulose membranes. Membranes were blocked with TTBS buffer (20 mM Tris, pH 7.4, 150 mM NaCl, and 0.05% Tween 20) containing 5% nonfat dry milk for 1 h and were washed with TTBS buffer. For the detection of apoptotic and antiapoptotic proteins, nitrocellulose membranes were incubated in the antibodies to cytochrome c (BD Transduction Laboratories, Lexington, KY), PARP1 (catalog no. SC-1562), Bcl-2 (catalog no. SC-492), Bax (catalog no. SC-426), Bak1 (catalog no. SC-832), Bid (catalog no. SC-6291), Bad (catalog no. SC-943), TRP53 (catalog no. SC-100), and caspase-8, specific for cleaved caspase-8 (catalog no. SC-7890) (Santa Cruz Biotech, Santa Cruz, CA), caspase-3 (catalog no. SC-9961), caspase-9 (catalog no. SC-9506), and caspase-10 (catalog no. SC-9752) (Cell Signaling Technology, Beverly, MA). After exposure to horseradish peroxidase-conjugated anti-rabbit IgG (caspase-3, caspase-8, caspase-9, caspase-10, Bak1, and Bcl-2), anti-goat IgG (Bid and PARP1), or anti-mouse IgG (cytochrome c, TRP53, and Bax), secondary antibodies (diluted 2000-fold to 5000-fold) for 1 h, blots were washed and developed by enhanced chemiluminescence (ECL kits; Amersham Life Science, Piscataway, NJ). Each blot was stripped with 100 mM glycine, pH 2.3, and was reprobed with ß-tubulin or heat shock protein 1 (HSPD1) to normalize for any variations incorporated in protein loading. Densities of each protein of interest were expressed as a ratio to that of ß-tubulin or HSPD1 on the same blot.
Analysis of the placental and fetal weights was performed by taking the mean of the placental and fetal weights per rat first, followed by the mean of all the rats. Weights are expressed as mean ± SEM and are compared using 1-way ANOVA for comparing various doses of ADM2 antagonist. Statistical analysis between the two groups was performed using the Student t-test. Values were considered significant at P < 0.05.
Effects of ADM217–47 on Fetoplacental Weights
In this study, we evaluated the role of ADM2 in the regulation of fetoplacental growth during pregnancy. ADM217–47, an antagonist of ADM2, was continuously infused to pregnant rats through osmotic minipumps beginning on Gestational Day 15. These animals received 50 or 200 µg/day of ADM217–47 or of vehicle only and were killed on Day 18 of gestation to assess placental and fetal weights. Figure 1, A and B, shows that fetal and placental weights in rats receiving the two doses of ADM2 antagonist were significantly lower than those in controls (P < 0.05). The reductions in placental weights were more substantial with ADM217–47 at 200 µg/day compared with 50 µg/day showing 11% decline compared to the controls. Fetal weights follow a trend of decreasing with increasing doses of ADM217–47 showing 17% decline with higher dose compared to controls; however, the changes observed in the fetal weights were not significantly different between the two doses of ADM217–47. Because the effects of ADM217–47 on the placenta were more substantial at 200 µg/day, we used this dose in all our subsequent studies.
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Morphological Changes in Rat Placenta Associated with ADM217–47 Infusion
We used H&E staining to examine ADM217–47-associated changes in rat placental sections obtained on Day 18 of gestation from ADM217–47-treated and vehicle alone-treated rats. As shown in Figure 2, ADM217–47 infusion caused impairment of labyrinths and distortion of decidua indicative of deficient placental vasculature. These morphological changes observed in the ADM217–47-treated animals suggest a role for ADM2 in placental angiogenesis in rat pregnancy.
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Effects of ADM217–47 on NOS2 and NOS3 Transcripts in Placenta
NO is implicated as a bioactive molecule regulating vascular function, platelet aggregation, neurotransmission, immunological function, and embryo implantation [19–22]. Trophoblasts express at least two isoforms of NOS enzyme, NOS2 and NOS3 [23]. To assess whether ADM217–47 infusion alters the NO system in placenta, we examined the changes in transcript levels for NOS2 and NOS3 in placental tissues of ADM217–47-treated and vehicle-treated animals. As shown in Figure 3, the transcript levels for NOS2 and NOS3 are significantly lower in placentas from ADM217–47-treated rats compared with the controls.
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Apoptotic Changes in Placental Tissues with ADM217–47 Infusion in Pregnant Rats
Because fetal growth and placental weights were significantly lower in ADM217–47-treated rats, we examined the possibility of involvement of apoptosis in the observed ADM217–47-associated reduction in placental and fetal growth. Apoptosis is demonstrated within the placental tissue by the TUNEL method. Most TUNEL-positive apoptotic cells were trophoblasts and were primarily found in the labyrinth and the junctional zone in placenta. The number of TUNEL-positive cells per microscopic field at 100x magnification was 205 ± 9.4 in the labyrinth zone of the ADM217–47-treated animals compared with 111 ± 2.8 in the control animals. The number of TUNEL-positive cells per microscopic field at 100x in the junctional zone was 54 ± 4.5 in the ADM217–47-treated animals compared with 9 ± 0.5 in the control animals. Therefore, there was an increase in the number of cells with TUNEL-positive staining in the labyrinth and in the junctional zone in the ADM217–47-treated animals compared with untreated control rats (Fig. 4). Specificity of staining was demonstrated by the absence of staining when Dut was eliminated from the staining protocol.
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Effects of ADM217–47 on the Expression of the Bcl-2 Family of Proteins in Placenta
The Bcl-2 family of proteins plays an important role in the regulation of apoptosis in a variety of cells. To study the pathways involved in ADM217–47-induced apoptosis, we examined the changes in proapoptotic and antiapoptotic Bcl-2 family proteins. Figure 5 shows the changes associated with ADM217–47 treatment in Bcl-2, Bax, Bak1, Bad, and Bid proteins in placenta. Significant decreases in the expression of the antiapoptotic Bcl-2 protein in placenta were observed in the ADM217–47-treated group compared with the controls. Expression of the proapoptotic proteins Bad and Bak1 was significantly elevated in the placenta of treated animals compared with the control animals, whereas there were no significant ADM217–47-associated changes in the expression of the proapoptotic proteins Bax and Bid.
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Changes in Mitochondrial Cytochrome c Associated with ADM217–47 Treatment
Release of cytochrome c from mitochondria is a critical component in the intrinsic apoptotic process. It occurs upstream of caspase activation and is inhibited by Bcl-2 overexpression. We measured cytochrome c content in mitochondrial and cytosolic fractions of placental tissues from control and ADM217–47-treated rats. As shown in Figure 6, Western blot analysis revealed that cytochrome c levels are significantly lower in mitochondrial fraction and are elevated in the cytosol (P < 0.05) of ADM217–47-treated rats compared with untreated rats, indicating an increase in the release of cytochrome c from mitochondria.
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Involvement of Caspase-3 Activation in ADM217–47-Induced Apoptosis
Caspases are the key executioners of apoptosis and cell death. In this study, we identified involvement of the caspase family of proteases in ADM217–47-induced apoptosis. Because caspase-3 is a central effector of apoptosis, we examined the proteolytic processing of caspase-3 and its downstream target, PARP1 protein, by Western blot analysis. Consistent with cytochrome c efflux from mitochondria, ADM217–47 treatment increased the proteolytically cleaved (active) caspase-3 fragments in the placenta of ADM217–47-treated animals compared with controls. In addition, we identified the involvement of caspase-9, because it is also known that mitochondrial dysfunction results in cytochrome c release with subsequent activation of caspase-9, one of the major caspase initiators. Active caspase-9 protein levels in the placenta of ADM217–47-treated animals were significantly (P < 0.05) higher compared with those of control animals (Fig. 7). Furthermore, we observed a trend of increasing levels of cleaved fragments of PARP1 in placentas with ADM217–47 treatment; however, the increase was not significant. These data provide evidence for the involvement of the mitochondria-related apoptotic pathway in ADM217–47-induced apoptosis in the rat placenta.
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Caspase-8, Caspase-10, and TRP53-Mediated Signaling Does Not Contribute to ADM217–47-Induced Apoptosis
Because caspase-8 and caspase-10 are activated as part of an extrinsic apoptotic pathway downstream to Fas/Fas stimulation, we examined caspase-8 and caspase-10 levels in placentas of ADM217–47-treated and control rats. Figure 8 shows no significant differences in caspase-8 and caspase-10 in ADM217–47-treated placenta compared with controls. To study the contribution of TRP53 in ADM217–47-induced apoptosis, we examined TRP53 protein levels in placental tissues in ADM217–47-treated rats and control rats. There were no differences in TRP53 levels in placentas between treated and control groups (Fig. 8).
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CALCA and ADM are expressed in placenta and have been shown to play a role in maintaining normal placental function. In the present study, we demonstrated for the first time (to our knowledge) that ADM2, a novel member of the calcitonin/CALCA family of peptides, has a role in pregnancy and is important for fetoplacental growth and development. Infusion of ADM217–47 to pregnant rats on Day 15 caused significant decreases in fetal and placental weights. In addition, we identified decreases in NOS3 and NOS2 expression with enhanced apoptotic changes in the mitochondrial-related apoptotic pathway proteins in the ADM217–47-treated placenta. Furthermore, impaired morphological changes in the labyrinth and the decidua of the treated placenta were also observed. Therefore, we suggest a role for endogenous ADM2 in the regulation of placental function and fetal development during pregnancy.
Levels of ADM2 in rat plasma are higher than levels of ADM in the rat [11]. However, there are no reports on the regulation of circulatory ADM2 in pregnancy, to our knowledge. Levels of CALCA and ADM in the maternal plasma are elevated during pregnancy in humans and rats [24, 25]. In pathological pregnancies, ADM concentrations in the maternal plasma and in fetal placental tissues mediate compensatory vascular responses in placental and fetal circulation [26]. ADM transcript levels are elevated in preeclampsia to mediate local fetal and placental adaptive response to reduced placental perfusion [27]. In Figure 1, we show that infusion of ADM217–47 to pregnant rats caused a significant decline in the fetal and placental weights, implicating that endogenous ADM2 is important for fetal and placental growth and development. Although both doses of ADM217–47 used in this study showed significant adverse effects on placental and fetal weights, the difference between the two doses (50 vs. 200 µg) was significant only in placenta. In similar investigations performed with different doses of ADM22–52 infused on Day 14 of gestation, blocking of endogenous ADM caused significant decreases in fetal and placental weights, but the effects observed were not dose dependent [14]. The reductions in placental and fetal weights in ADM217–47-infused rats are supported by the morphological changes observed. As shown in Figure 2, labyrinth and decidual morphology is impaired in the treated group compared with the controls. The labyrinth is primarily responsible for fetomaternal nutrient exchange; therefore, an impaired labyrinth as evidenced by the morphological changes and increased TUNEL-positive staining (Fig. 4) underlies the ADM217–47-induced fetal growth restriction. Distorted decidua observed in the ADM217–47-treated animals may result from the changes in maternal vascular function. Deficient vascular development has been shown in Adm gene knockout mice [28], and ADM is reported to be involved in inhibiting apoptosis and in promoting angiogenesis [29]. Morphological changes in the placenta observed in the ADM217–47-treated rats are indicative of deficient vasculature in the labyrinth. Therefore, these data suggest that ADM2 may be involved in placental angiogenesis during fetoplacental development.
Our unpublished data suggest that ADM2 is expressed in the rat placenta. Therefore, the effects of ADM217–47 on fetoplacental growth could be mediated through opposition to the actions of ADM2 expressed locally in the placenta or delivered through the circulation. Furthermore, in Figure 3, we illustrate the involvement of the NO system in the ADM2-induced effects in the placenta. NO is a bioactive molecule regulating vascular and immunological functions [30]. Previous investigations show that NO may play a role in placental function and that intrauterine injection of NOS inhibitor during the periimplantation period decreases the embryo implantation rate in rats [31], Furthermore, early embryos can generate NO [32], and trophoblasts express NOS3 and NOS2 [23, 33]. Because the ADM2 effects are mediated through NO [8, 18], we analyzed the expression profiles of NOS3 and NOS2 transcripts in the rat placenta treated with 200 µg/day of ADM217–47. As shown in Figure 3, significant decreases were observed in both isoforms of NOS in the placentas of treated rats compared with controls. Although changes in transcript levels do not necessarily reflect protein levels or the activity of these enzymes, this data indicate that the effects of ADM2 on the placenta may involve the NO system. NO is suggested to modulate apoptosis in the microvasculature [34], and reduced expression of the NOS isoforms may underlie the vascular insufficiency observed in the ADM217–47-treated rats (Fig. 2).
Antagonizing the ADM receptors causes apoptosis in the rat placenta [15]. Because ADM2 acts through CALCA and ADM receptors, we hypothesized that the effects of ADM217–47 on placental growth restriction and impaired placental morphology observed in the treated placenta may also involve enhanced apoptosis. Indeed, ADM217–47 induced an increase in TUNEL-positive staining in the placenta as shown in Figure 4. The staining was more predominant in the labyrinth and the junctional zone, indicating that ADM217–47-induced apoptotic changes in the labyrinth and the junctional zone seem to play a role in the observed fetal and placental growth restriction.
Because CALCRL in association with RAMPs is a receptor for the three ligands CALCA, ADM, and ADM2, one cannot rule out the possibility that some of the observed ADM217–47 effects in rat placenta could be due to ADM217–47 blocking the effects of ADM or CALCA. Although CALCA antagonist completely blocks CALCA and ADM effects, it partially blocks ADM2 effects, whereas ADM217–47 can completely block ADM2 effects [1]. In addition, recent investigations on CALCRL knockout mice demonstrate that disruption of the CALCRL gene is lethal to embryonic development [35], suggesting the importance of its ligands in fetoplacental function. However, further studies are needed to assess the effects of ADM217–47 on ADM and CALCA functions.
Apoptosis is a physiological process that is highly orchestrated at the molecular level by the activation of an aspartate-specific cysteine protease (caspase) cascade. There are three leading factors triggering apoptosis: 1) increase in apoptosis-inducing genes, 2) suppression of apoptosis-inhibiting genes, and 3) enhanced calcium intake by the cell [36, 37]. There are two reported pathways leading to activation of caspases. The first is the mitochondrial (receptor independent) pathway, and the second involves interaction of the death receptor and its ligand [36, 38]. Proapoptotic and antiapoptotic Bcl-2 family members regulate mitochondrial pathways by regulating cytochrome c release from the mitochondria. Oligomerization of apoptotic protease activating factor 1 by the cytosolic cytochrome c results in activation of caspase-9, which then activates procaspase-3. On the other hand, the activation of Fas causes activation of caspase-8, which then can activate procaspase-3 directly or through cleaving proapoptotic protein, Bid, which subsequently induces cytochrome c release from mitochondria.
The present study provides evidence that infusion of ADM217–47 activates the mitochondrial apoptotic pathway in the placenta. This conclusion is supported by several observations. As shown in Figure 5, ADM217–47 infusion caused significant decreases in Bcl-2 protein expression, with a reduction in the mitochondrial cytochrome c content in the placenta. Furthermore, we demonstrate elevated levels of caspase-9 and caspase-3 in the ADM217–47-treated placenta (Fig. 7). In this study, we observed decreased levels of mitochondrial cytochrome c in ADM217–47-treated placenta, with an increase in the cytosolic fraction (Fig. 6). However, it is unclear from this study how ADM217–47 infusion triggers the release of mitochondrial cytochrome c. Bcl-2 belongs to a growing family of apoptosis regulatory gene products that may be antiapoptotic (Bcl-2, BclXL, Bcl-2L2, Bcl-2A1A, BRAG1, and MCL1) or proapoptotic (Bax, Bak1, Bad, Bid, Bik, and BclXS). We observed a significant increase in the levels of proapoptotic proteins Bad and Bak1 in the placenta of ADM217–47-treated animals. Fas and Fas ligand signaling constitutes the extrinsic apoptotic pathway involving activation of downstream effector proteins like Bid, caspase-8, and caspase-10. In this study, we observed no change in the expression profile of Bid (Fig. 5), suggesting that the extrinsic pathway leading to caspase-8-dependent cleavage of Bid is not critical to ADM217–47-induced apoptosis in rat placenta. This is further supported by Figure 8, which shows no change in caspase-8 or caspase-10 expression in ADM217–47-treated rat placenta. Expression levels of TRP53 proteins are elevated in response to DNA damage in apoptosis. The Bax promoter region has a binding site for TRP53; therefore, we analyzed whether ADM217–47 has any effect on the levels of TRP53. Our findings exclude the involvement of TRP53, a mediator of extrinsic and intrinsic apoptotic pathways and a key player in cellular responses to stress and cell proliferation in ADM217–47-induced apoptosis.
Downregulation of Bcl-2, cytochrome c release, and activation of caspase-3 observed in ADM217–47-treated placenta implicate involvement of the intrinsic pathway in ADM217–47-induced apoptosis in rat placenta. Apoptosis is a physiological process and plays a leading role in tissue embryogenesis and development. Enhanced apoptosis may be involved in the development of widespread diseases, including preeclampsia and intrauterine growth retardation. This study demonstrates that infusion of ADM2 antagonist causes fetoplacental growth restriction through placental apoptosis by activation of the intrinsic pathway. Findings from our study also suggest possible involvement of the NO system in ADM217–47-induced effects on rat fetal and placental growth. Although the present study does not define a precise mechanism of apoptosis induced by ADM217–47 in rat placenta, the results suggest a role for ADM2 in maintaining normal placental function and fetal growth.
ACKNOWLEDGMENTS
We thank Cheryl Welch for her administrative assistance.
FOOTNOTES
1Supported by grants HL-58144 and HL-72650 from the National Institutes of Health. 
Correspondence: 2 Chandrasekhar Yallampalli, Department of Obstetrics and Gynecology, 301 University Boulevard, MRB, 11.138, Route 1062, Galveston, TX 77555-1062. FAX: 409 747 0475; e-mail: chyallam{at}utmb.edu
Received: 24 April 2006.
First decision: 2 June 2006.
Accepted: 27 August 2006.
REFERENCES
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