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Original Studies |
Department of Reproductive Medicine and Child Development, Division of Obstetrics and Gynecology, University of Pisa (T.S., M.S., A.R.G.), and Scuola Superiore di Studi e di Perfezionamento S. Anna (T.S.), Pisa; the Institute of Obstetrics and Gynecology, Catholic University (R.A., F.M.), Rome; OASI Institute of Research (A.L.), Troina; and the Department of Surgery, Chair of Obstetrics and Gynecology University of Udine (F.M.R., L.D., F.P.), 33100 Udine, Italy
Address all correspondence and requests for reprints to: Felice Petraglia, M.D., Department of Surgery, Chair of Obstetrics and Gynecology, University of Udine, 33100 Udine, Italy. E-mail: felice.petraglia{at}dsc.uniud.it
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Abstract |
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were potent stimulators of CRF release from HUVEC.
Among the peptides, CRF secretion was stimulated by interleukin-1ß
and by endothelin-1. Our study shows for the first time that HUVEC express CRF messenger ribonucleic acid and peptide as well as the CRF R2 gene, and that CRF release is differentially regulated by several distinct molecules. We here propose that CRF has a role in the regulation of the fetal-placental circulation.
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Introduction |
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Besides representing an important endocrine modulator, CRF is a potent vasoactive molecule. Indeed, in different animal species CRF has been shown to induce vasodilatation on various vascular beds (4, 5, 6) by inducing vascular smooth muscle cells relaxation (7). The vasoactive properties of CRF may be of great physiological importance during human pregnancy, and the feto-placental circulation may directly represent a target for CRF. In fact, CRF infusion in perfused human placental lobules triggers a potent vasodilatation (8), and this effect appears to be mediated through a nitric oxide-cGMP pathway (9), suggesting the involvement of vascular endothelium as a mediator of the effect of CRF.
Additional indirect suggestions of a potential importance of CRF in the control of human feto-placental circulation are provided by the evidence that pregnancies complicated by preeclampsia and intrauterine growth retardation are associated with abnormal placental vascular resistance and blood flow (10) and with abnormally high umbilical vein CRF levels (11, 12).
As the endothelium is a critical regulator of vascular function in both physiological and pathological conditions, we investigated whether cultured human umbilical vein endothelial cells (HUVEC) may represent a source and a possible target for CRF. CRF messenger ribonucleic acid (mRNA) expression was assayed on cultured HUVEC by reverse RT-PCR gene expression analysis and by detecting immunoreactive CRF in culture medium. As type 2 CRF receptor as been shown to be expressed in other vascular beds, we studied by RT-PCR its possible expression in HUVEC. Finally, the regulation of CRF release from HUVEC by various substances was assessed using a specific RIA.
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Materials and Methods |
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Human umbilical cords were obtained from healthy women who underwent uncomplicated term pregnancies (n = 11). Informed consent was obtained from each subject. After collection, the umbilical cord was rapidly immersed in sterile saline solution (0.9% NaCl) and immediately processed for endothelial cell isolation. Under a laminar flow sterile hood, the umbilical vein was cannulated and thoroughly rinsed with sterile saline solution. After clamping the other extremity, the vein was filled for 10 min with 0.2% type IA collagenase (Sigma Chemical Co., Inc., St. Louis, MO) solution (in HBSS without Ca2+ and Mg2+) prewarmed at 37 C. The collagenase solution was then discarded, and the vein was gently washed with 40 mL HBSS (Ca2+ and Mg2+ free) that was collected in a 50-mL sterile polypropylene tube. The collected cells were pelleted by centrifugation at 4 C for 10 min at 1300 rpm, the supernatant was discarded, and the cell pellet was gently resuspended in medium 199 (Life Technologies, Inc., Gaithersburg, MD) containing 20% heat-inactivated fetal bovine serum (HyClone Laboratories, Inc. Logan, UT), antibiotics, endothelial cell growth factor (20 µg/mL), and heparin (50 µg/mL). The cells were then plated on culture plates precoated with 0.5% sterile gelatin (Sigma Chemical Co., Inc.). The endothelial cell identity was confirmed by the typical cobblestone aspect and by immunostaining with an antibody vs. factor VIII-related antigen in representative dishes. Once grown to confluence, the cells were replated on gelatin-coated culture dishes at 20,000 cells/cm2 and grown up to the moment of the experiments.
Total RNA extraction and quantitation
To collect total RNA (for RT-PCR), cells were plated in 55-cm2 culture dishes. Once grown to confluence, the cells were immediately treated for total RNA extraction. Total RNA was obtained by the standard acid guanidinium thiocyanate-phenol-chloroform extraction method (13) and was then quantified spectrophotometrically by UV absorption at 260 nm. Aliquots of the total RNA were checked by electrophoresis on a 1.2% agarose gel, and ethidium bromide staining of the 18S and 28S ribosomal RNA subunits was used to confirm the absence of degradation and/or genomic contamination.
RT-PCR
RT-PCR experiments were carried out according to the GenAmp amplification reaction kit instructions provided by the manufacturer (Perkin Elmer Corp., Milan, Italy). Total RNA (1 µg) was reverse transcribed to complementary DNA (cDNA) by a reaction containing 1 mmol/L each deoxynucleotide triphosphate, 1 U ribonuclease inhibitor, 100 pmol random hexamers, and 200 U murine Moloney virus reverse transcriptase. The reaction was run at 42 C for 1 h, heated at 94 C for 5 min, and then quickly chilled on ice. CRF and CRF R2 cDNAs PCR amplifications were carried out by the use of two specific oligonucleotide primer sets synthesized on the basis of the published CRF and CRF R2 gene sequences (CRF sense primer, 5'-TCCGAGGAGCCTCCCATC-3'; CRF antisense primer, 5'-AATCTCCATGAGTTTCCTGTTGC-3'; GenBank accession no. V00571; CRF R2 sense primer, 5'-TCGTCAACTACCTGGGCCAC-3'; CRF R2 antisense primer, 5'-GTCATTAGGATCCTGACGATGT-3'; GenBank accession no. U34587). Computer analysis performed to compare the synthesized oligomers to the published human sequences with Basic Local Alignment Search Tool (BLAST) software revealed no significant homology toward any of all the other known human gene sequences. CRF cDNA amplification was performed with 30 thermal step cycles, during which the reaction mixture was heated at 94 C for 1 min, cooled at 60 C for 25 s, and then heated at 72 C for 1 min and 10 s. CRF R2 cDNA amplification was performed with 35 thermal step cycles programmed as follows: 94 C for 1 min, 55 C for 1 min, and then 72 C for 3 min. As positive controls during CRF and CRF R2 RT-PCRs, parallel reverse transcribed and PCR-amplified 1 µg total RNAs extracted either from third trimester human placental trophoblast, which is known to abundantly express CRF gene (14), or from third trimester gestational human myometrium, which hat has been shown to express CRF R2 (15), were used in every experiment. The negative control was a blank prepared using all reagents and substituting an equal volume of bidistilled sterile water for total RNA. The negative samples revealed no amplification products. In addition, possible genomic DNA contaminations were ruled out by parallel RT-PCRs performed without adding murine Moloney virus reverse transcriptase to the reactions. These control reactions constantly produced no detectable amplified products. Amplification products were run on a 4% agarose gel stained with ethidium bromide and photographed on a UV transilluminator. The experiments were repeated three times, using RNAs extracted from cell cultures derived from six different umbilical cords, and qualitatively similar results were obtained.
CRF assay
To measure immunoreactive CRF (irCRF) secreted in culture
medium, cells were plated in 24-well culture dishes, grown to
monolayers (corresponding to 
5 x 105 cells), and
then incubated according to the different experimental conditions in 1
mL culture medium. In particular, cells were incubated for 0, 2, 4, 6,
or 24 h in fresh medium without any addition to assess basal irCRF
time-dependent secretion. In parallel, other cells were incubated for
4 h in the presence of forskolin (10-6 mol/L),
PGF2 (10-6 mol/L), dexamethasone
(10-5 and 10-6 mol/L), progesterone
(10-6 mol/L), 17ß-estradiol (10-6 mol/L),
interleukin-1ß (IL-1ß; 1 and 10 µg/mL), or endothelin-1
(10-6 mol/L). At the end of each experiment, culture media
were collected in glass tubes with aprotinin (as a protease inhibitor)
and then immediately frozen at -20 C, where they remained until the
assay. Four different wells were used for each experimental condition,
and culture media were separately collected and assayed for irCRF
detection.
Before the assay, all samples were dried in a speed vacuum concentrator (Savant Instrument Co., Hicksville, NJ), and each sample was redissolved in assay buffer (100 mmol/L NaCl, 50 mmol/L NaPO4, 25 mmol/L ethylenediamine tetraacetate, 0.1% BSA, 0.05% Triton X-100, and 0.1% sodium azide, pH 7.3). CRF concentrations were measured in duplicate. Rabbit antihuman CRF antibody was used at a final dilution of 1:600,000 (W. Vale, The Salk Institute, La Jolla, CA). [I125]CRF (Amersham International, Aylesbury, UK) was used as a tracer as previously described (16). Synthetic human CRF (J. Rivier, The Salk Institute) was used to prepare the standard curve.
The sensitivity of the CRF RIA was 2.5 pg/mL; water blanks gave a constant binding over 97.5%, which is equal to 0. The inter- and intraassay coefficients of variation were 8% and 4%, respectively. All assays were repeated three times, using cells from three different patients, with similar results.
Statistical analysis
All values are expressed as the mean ± SD. Multiple comparisons were performed by one-way ANOVA, and individual differences were tested by the Fisher’s protected least significance difference (PLSD) test after the demonstration of significant intergroup differences by ANOVA. Two-group comparisons were performed by unpaired Student’s t test.
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Results |
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RT-PCR experiments performed on total RNA extracted from cultured
HUVEC revealed the presence of CRF mRNA expression (Fig. 1
). Endothelial cell samples used for
RT-PCR amplifications produced a clear and intense 122-bp band on
ethidium bromide-stained agarose gel, corresponding to the expected
length (Fig. 1). Total RNA samples extracted from third trimester human
trophoblast were used as positive controls (Fig. 1), and the amplified
products from these samples corresponded in length to those amplified
from the endothelial cells. Negative controls (performed as detailed in
Materials and Methods) provided evidence of the absence of
genomic contamination.
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irCRF concentrations were assayed in culture medium from untreated
confluent endothelial cells at different incubation time points. irCRF
was detectable only at very low levels at the beginning of the
incubation (Fig. 2). After 2 h,
irCRF levels significantly increased and subsequently remained stable
at 4 and 6 h of incubation, with a significant decrease after
24 h of incubation (Fig. 2).
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Cultured HUVEC also express CRF receptor type 2 mRNA, thus
potentially representing one of the targets of CRF itself at the
vascular level (Fig. 3). Myometrium from
pregnant women was used as a positive control (Fig. 3).
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The adenylate cyclase activator forskolin produced a clear
stimulation of CRF release from HUVEC (Fig. 4A). Treatment with IL-1ß was similarly
able to produce a dose-related stimulation of CRF secretion (Fig. 4A)
from cultured endothelial cells. Incubation of HUVEC in the presence of
dexamethasone significantly reduced CRF production (Fig. 4B), showing
an effect similar to that exerted at the hypothalamic level.
Progesterone and 17ß-estradiol, at concentrations similar to those
found in the third trimester of pregnancy, induced a strong increase in
CRF immunoreactivity in the culture medium (Fig. 4C). The
vasoconstrictor endothelin-1 induced an increase in CRF secretion by
endothelial cells (Fig. 4D), and the potent vasoactive
PGF2 strongly increased CRF concentrations as well (Fig. 4D).
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Discussion |
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CRF expression in umbilical vein endothelial cells appears to be positively regulated by cAMP, as demonstrated by the stimulatory effect exerted by forskolin, an activator of adenylate cyclase. The activation of CRF production through the cAMP-dependent pathways is a common feature in other CRF-expressing tissues (17) and correlates with the described presence of a transcriptionally active perfect cAMP-responsive element on the 5'-flanking sequence of the CRF gene (17). The presence of a similar mechanism in endothelial cells suggests that the regulation of the promoter region by cAMP-dependent transcription factors may be important for the control of CRF gene expression at this level also.
The regulation by a classical proinflammatory cytokine such as IL-1ß suggests a CRF regulation, as in human placenta (16), and a possible involvement for CRF at the endothelial level in the local regulation of the immune-inflammatory processes may be hypothesized. In fact, previous evidence clearly demonstrates that CRF is a peripheral autocrine/paracrine regulator of these responses in several tissues (18). Vascular endothelium is a critical controller of inflammation, particularly through the regulation of lymphocyte-leukocyte extravasation, that is triggered by inflammatory cytokines such as IL-1 (19), and the cross-talk between cytokines and CRF may potentially represent a novel mechanism for the local control of the immune-inflammatory responses by the vascular endothelium.
Similar to other biological systems, steroid hormones modulate CRF release from endothelial cells. 17ß-Estradiol and progesterone have a potent stimulatory effect on CRF secretion. Indeed, under the stimulatory effect of these molecules, the CRF concentration in the culture medium increased 20–30 times, reaching secretion rates in the same range as those that characterize the human placenta and the fetal membranes at term pregnancy (20). The human CRF gene has been demonstrated to be under direct regulation by 17ß-estradiol (21). The human CRF gene promoter contains five putative half-palindromic consensus elements for the estrogen receptors (17), and 17ß-estradiol has been shown to increase the activity of reporter constructs corresponding to the CRF gene promoter region during transfection experiments via a specific association between the estrogen-responsive elements and the DNA-binding domain of the human estrogen receptor (21). These data confirm our results and suggest that the stimulatory action of estradiol on CRF expression in endothelial cells may be linked to a transcriptional effect.
In contrast to CRF release from human placenta (20, 22), glucocorticoids reduced CRF output from endothelial cells, thus suggesting the existence of a gene regulation similar to that present at the hypothalamic level (17). The fact that a pharmacological dose of dexamethasone was required to significantly inhibit basal CRF release may be due to the obvious limitations of the in vitro system and does not conflict with a possible physiological relevance of lower levels of the molecule.
Similarly, progesterone decreases CRF production from cultured placental cells (20), but stimulates CRF secretion on umbilical vein endothelial cells. During pregnancy, increasing concentrations of progesterone and 17ß-estradiol may progressively increase the local production of CRF in the umbilical vein, thus potentially increasing blood flow in the feto-placental circulation via the vasodilatory action of this peptide. In fact, previous studies have shown that CRF possesses a potent vasodilatory action on the human feto-placental circulation, being more active than prostacyclin in inducing vasodilatation, with an action involving a nitric oxide-mediated pathway (8, 9).
Additional evidence of a prospective role for CRF as an endothelial
regulator of vascular tone is provided by the present finding of CRF
release induction by two potent vasoconstrictors, PGF2
and ET-1, thus suggesting a role for CRF in the dynamic loop between
vasodilators and vasoconstrictors.
Although the expression of a mRNA does not necessarily reflect the presence of the biologically active encoded protein, the demonstration by RT-PCR of CRF receptor type 2 mRNA in umbilical vein endothelial cells is compatible with a possible action of CRF as a direct modulator of umbilical vein homeostasis during pregnancy, suggesting a potential autocrine/paracrine activity.
Recently, a marked vasodilatory activity on the fetal-placental
circulation has been described also for a newly discovered member of
the CRF family: urocortin (23). This peptide is also expressed in the
feto-placental unit (24) and binds with high affinity to CRF receptor
type 2. The vasodilatory activity of urocortin was significantly
attenuated by the CRF receptor antagonist -helical CRF-(9–41) (23),
suggesting that the CRF receptor 2 may be involved in the effect of the
peptide. This finding perfectly fits with our demonstration of the
expression of the CRF type 2 receptor’s mRNA on endothelial cells, and
suggests that the endothelium may be a mediator of the vasodilatory
actions of both CRF and urocortin.
Therefore, our present data may help to explain the increased secretion of CRF in some pathological conditions. Indeed, maternal CRF concentrations are abnormally elevated in women with preeclampsia (25), pregnancy-induced hypertension (26), or preterm labor (27), and high CRF levels in the umbilical cord during pregnancies complicated by preeclampsia (11) or intrauterine growth retardation (12) have been described. Both conditions are associated with increased resistance to blood flow in the fetal-placental circulation (10), and the vasodilatory action of exogenous CRF appears to be impaired in placentas from these pathological conditions (9). These findings suggest that the increased CRF production may be involved in the adaptative response of the cardiovascular function to the hemodynamic derangement that characterizes these pathological status, and that in view of our results, endothelial CRF production may have a role in this process.
In summary, our study demonstrates for the first time that human endothelial cells express CRF and a CRF receptor mRNA, and that CRF production from endothelial cells is differently regulated by several molecules. These findings suggest that locally produced CRF may represent a novel, as yet unknown, endothelial modulator and that CRF may directly regulate the human fetal-placental circulation at the vascular level, by exerting a regulatory role on umbilical vein endothelial cells during pregnancy.
Received February 12, 1999.
Revised April 5, 1999.
Accepted April 15, 1999.
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