Dominique Cabrol

PDE4 inhibition prevents preterm delivery induced by an intrauterine inflammation.

The aim of this study was to explore the anti-inflammatory properties of phosphodiesterase-4 (PDE4) inhibitors in vivo and their potential ability to prevent inflammation-induced preterm delivery. Indeed, intrauterine inflammation is the major etiology of very preterm delivery, the leading cause of neonatal mortality and morbidity. Intrauterine injection of Escherichia coli LPS in 15-day-pregnant mice induced an increase of PDE4 activity and PDE4B expression at the maternofetal interface, a rise of amniotic fluid levels of TNF-α, IL-1β, IL-6, and IL-10 and provoked massive preterm delivery and fetal demise. Selective PDE4 inhibition by rolipram prevented the rise in the proinflammatory cytokines. Following the nuclear translocation of the transcription factor NFκB, as a marker of cellular activation after the inflammatory challenge, showed a time-dependent sequential activation of the gestational tissues, from the uterine mesometrial to the fetal compartment, particularly in the glycogen-trophoblastic cells of the placenta. This activation was disrupted by PDE4 inhibition, and inflammation-induced preterm delivery and fetal demise were prevented. PDE4 selective inhibitors may thus represent a novel effective treatment to delay inflammation-induced preterm delivery and to prevent adverse outcomes in infants.

Evidence for a Role of Phosphodiesterase 4 in Lipopolysaccharide-Stimulated Prostaglandin E2 Production and Matrix Metalloproteinase-9 Activity in Human Amniochorionic Membranes

Chorioamniotic infection is a leading cause of preterm premature rupture of fetal membranes (amnion and chorion). Bacterial infection induces an inflammatory response characterized by elevated production of proinflammatory cytokines; the latter activate the production of both PGs that stimulate uterine contractions, and matrix metalloproteinases (MMPs) that degrade the extracellular matrix of the chorioamniotic membranes. The inflammatory response is under the control of cAMP content, which is partly regulated by phosphodiesterases (PDE). In this study, we investigated the role of the PDE4 family in the inflammatory process triggered by LPS in a model of amniochorionic explants. We found that PDE4 family is the major cAMP-PDE expressed in human fetal membranes and that PDE4 activity is increased by LPS treatment. Selective inhibition of PDE4 activity affected LPS signaling, because PDE4 inhibitors (rolipram and/or cilomilast) reduced the release of the proinflammatory cytokine TNF-α and increased the release of the anti-inflammatory cytokine IL-10. PDE4 inhibition reduced cyclooxygenase-2 protein expression and PGE2 production and also modulated MMP-9, a key mediator of the membrane rupture process, by inhibiting pro-MMP-9 mRNA expression and pro-MMP-9 activity. These results demonstrate that the PDE4 family participates in the regulation of the inflammatory response associated with fetal membrane rupture during infection. The PDE4 family may be an appropriate pharmacological target for the management of infection-induced preterm delivery.

Selective Protein Kinase C Isoforms Are Involved in Endothelin-1-Induced Human Uterine Contraction at the End of Pregnancy

Abstract The role of protein kinase C (PKC) in contraction of the human myometrium induced by endothelin-1 (ET-1) was investigated at the end of pregnancy. The expression and subcellular distribution of PKC isoforms were examined by Western blot analysis using isoform-specific antibodies. At least three conventional PKC isoforms (cPKC; α, β1, and β2), two novel PKC isoforms (ε and δ), and an atypical PKC isoform (ζ) were detected in pregnant myometrium. Quantitative immunoblotting revealed that all these isoforms were mainly distributed in the particulate fraction. The lack of a calcium chelator to modify the particulate sequestration of cPKC suggests an interaction with an anchoring protein such as receptor-activated C kinase-1, which is evidenced in the particulate fraction of the pregnant myometrium. Of the six isoforms, only PKCβ1, PKCβ2, PKCδ, and PKCζ were translocated to the particulate fraction, and PKCε to the cytoskeletal fraction, after stimulation with ET-1. Involvement of PKC in the ET-1-induced contractile response is supported by the inhibition caused by the PKC inhibitor calphostin C. However, we demonstrated that the selective cPKC isoform inhibitor, Gö 6976, as well as the substantial depletion of PKCβ1 and PKCε and the partial depletion of PKCα and PKCδ by a long-term treatment with phorbol 12,13-dibutyrate did not prevent ET-1-induced contraction. Accordingly, our results suggest that PKCδ and PKCζ activation mediated ET-1-induced contraction, whereas cPKC isoforms were not implicated in the human pregnant myometrium.

The PDE4 inhibitor rolipram prevents NF-kappaB binding activity and proinflammatory cytokine release in human chorionic cells

Spontaneous preterm delivery is linked to intrauterine inflammation. Fetal membranes are involved in the inflammatory process as an important source of mediators, and the chorion leave produces high levels of the proinflammatory cytokine TNF-α when stimulated by LPS. The transcription factor NF-κB is the main regulator of this inflammatory process and controls the production of cytokines by the chorion leave. Phosphodiesterase 4 inhibitors are recognized for their anti-inflammatory and myorelaxant effects. The purpose of this study was to investigate whether PDE4 inhibition affects the LPS signaling in human cultured chorionic cells. We showed that these cells express TLR4, the main LPS receptor, and exhibit a predominant PDE4 activity. Upon LPS challenge, PDE4 activity increases concomitantly to the induction of the specific isoform PDE4B2 and chorionic cells secrete TNF-α. LPS induces the nuclear translocation of the NF-κB p65 subunit and the activation of three different NF-κB complexes in chorionic cells. The presence of the PDE4 inhibitor rolipram reduces the TNF-α production and the activation of the three NF-κB complexes. These data indicate that the PDE4 family interacts with the LPS signaling pathway during the inflammatory response of chorionic cells. PDE4 selective inhibitors may thus represent a new therapeutic approach in the management of inflammation-induced preterm delivery.

Prostaglandin E2-induced changes in the distribution of glycosaminoglycans in the isolated rat uterine cervix

In order to study the hormonal control mechanisms of cervical ripening, we investigated the prostaglandin E2 (PGE2)-induced changes in the distribution of glycosaminoglycans (GAG) using hysterectomized and ovariectomized rats, leaving the vascularized uterine cervix in situ, as an animal model. In the first series of experiments, the GAG were measured in a control (n = 22 Wistar rats) and in a PGE2-treated group (n = 20 Wistar rats) without steroid supplementation. In the second series of experiments, the GAG were measured in a control (n = 19) and in a PGE2-treated group (n = 18) receiving estradiol and progesterone supplements. After PGE2 treatment in the two series of experiments, and despite being surgically isolated from the uterine corpus, the cervix was still able to undergo some of the structural changes associated with normal ripening (increased hydration and hyaluronic acid concentration). This suggests that PGE2, acting directly on the cervix, could be, at least in part, a modulator of biochemical events which underlie normal cervical maturation. The animal model described here seems to be suitable for studying the hormonal mechanisms of cervical ripening and the regulatory relationship between cervical maturation and myometrial contractility, which are probably subject to concordant endocrine regulation.

Pregnancy-related changes in the distribution of glycosaminoglycans in the cervix and corpus of the human uterus

We investigated the variations in the distribution of glycosaminoglycans (GAG) in the cervical, isthmic and corporeal stroma of the pregnant and non-pregnant human uterus. The GAG were measured in non-pregnant (n = 9) and pregnant (n = 14) cervices, in non-pregnant (n = 8) and pregnant (n = 6) uterine isthmus, and in non-pregnant (n = 12) and pregnant (n = 5) uterine corpus. Heparan sulfate is abundant in the isthmic and corporeal stroma. This has to be related to a larger content in blood vessels and muscular cells at this level than in the cervix. The distribution of hyaluronic acid and dermatan sulfate is the same in the cervix as in the corpus, but the amount of these GAGs is lower in the former, which could be due to a lower connective tissue content. Two of the main features of cervical maturation, increased hydration, and decreased dermatan sulfate concentration, were found also in the pregnant corporeal stroma. These results suggest that connective tissue maturation is not only a cervical phenomenon and could play a role in active and/or passive mechanical properties of the myometrium during late pregnancy and labor.

Stimulation of the ADRB3 Adrenergic Receptor Induces Relaxation of Human Placental Arteries: Influence of Preeclampsia

Abstract Preeclampsia, which complicates 3–8% of pregnancies, is one of the leading causes of neonatal morbidity and mortality. Its pathophysiology remains unclear. The aim of the present study was to investigate the presence and the role of β2- and β3-adrenergic receptors (ADRB2 and ADRB3, respectively) in human placental arteries and to assess the influence of preeclampsia on ADRB responsiveness. SR 59119A, salbutamol, and isoproterenol (ADRB3, ADRB2, and nonselective ADRB agonists, respectively) induced a concentration-dependent relaxation of placental artery rings obtained from women with uncomplicated or preeclamptic pregnancies. SR 59119A-induced relaxation was unaffected by the blockade of ADRB1 and ADRB2 by 0.1 μM propranolol but was significantly decreased by the blockade of ADRB1, ADRB2, and ADRB3 by 10 μM propranolol. Both SR 59119A and salbutamol were associated with a significant increase in cAMP production that was significantly inhibited by pretreatment with 0.1 μM propranolol only for salbutamol. SR 59119A-induced relaxation (Emax = 28% ± 5% vs. 45% ± 4%, respectively) and cAMP production (2.7 ± 0.5 vs. 4.9 ± 0.4 pmol/mg of protein, respectively; P < 0.01) were decreased in arteries obtained from preeclamptic compared to normotensive women. Both ADRB2 and ADRB3 transcripts were expressed at the same level between arteries from normotensive and preeclamptic women. Western blot analysis, however, revealed a decreased expression of the ADRB3 immunoreactive protein in arteries from preeclamptic compared to normotensive women. We suggest the presence of functional ADRB2 and ADRB3 in human placental arteries. Even if preeclampsia is associated with an impairment of the ADRB3 responsiveness, ADRB3 agonists may have future pharmaceutical implications in the management of pregnancy-related disorders.

PDE4 as a target in preterm labour

Cyclic nucleotide phosphodiesterases (PDE) are the enzymes catalyzing the hydrolysis and inactivation of the second messengers, cAMP and cGMP. Eleven PDE families are described to date, and selective inhibitors of some PDEs families are currently used in clinic for treating cardiovascular disorders, erectile dysfunction, and pulmonary hypertension. Isoforms of the PDE4 family are involved in smooth muscle contraction and inflammation. PDE4 selective inhibitors are currently in clinical trials for the treatment of diseases related to inflammatory disorders. Because of their myorelaxant properties, we first examined their expression in human myometrium and uncover an increased expression of one specific isoform, PDE4B2, in the near-term myometrium as compared to myometrium in the nonpregnant state. Using human myometrial cells in culture, we demonstrated that PDE4B2 can be induced by its own substrate, under the control of one of the major utero-contractile agonists, PGE2, itself upregulated by the proinflammatory cytokine IL-1β. Functionally, augmentation of global PDE4 activity decreases the ability of β-adrenergic agonists (the most commonly used tocolytic drugs) to inhibit myometrial contraction at the end of pregnancy and during pathophysiological situations, such as persistent intrauterine inflammation which is a major cause of very preterm delivery. Currently exploring the anti-inflammatory properties of PDE4 inhibitors in gestational tissues, we recently demonstrated the ability of these drugs to block a persistent inflammatory response of the foetal membranes in Humans and to prevent inflammation-driven preterm delivery and foetal demise in mice. These data open up a new therapeutical strategy to prevent inflammation-induced preterm delivery and its sequelae in very preterm infants.

Prognostic Value of Cervical Distensibility Index Measurement in the Outcome of Pregnancies with Threatened Premature Labor

Threatened premature labor remains a difficult diagnostic and therapeutic problem, and various clinical scores have been designed to evaluate its prognosis. This study presents the use of a cervicotonometer to measure cervical distensibility in 58 women hospitalized for threatened premature labor. This value will be used to determine whether preterm labor is preceded by preterm cervical maturation and thus to assess prognosis. The cervical distensibility index (CDI) obtained with the cervicotonometer was significantly elevated at the time of hospitalization in women who had a preterm delivery when compared to those who delivered at or after 37 weeks of amenorrhea, whereas clinical scores showed no difference between these two groups. No preterm deliveries occurred when the CDI was normal or low at the time of hospitalization. Therefore, CDI measurement, reflecting cervical maturation, seems to be a good prognostic indicator in premature labor.

Mechanical Properties of the Pregnant Human Uterine Cervix Use of an Instrument to Measure the Index of Cervical Distensibility

An instrument has been designed and manufactured to measure precisely in clinical practice the distensibility index of the human uterine cervix expressed as centimeters per kilogram. Operation of the instrument and possible errors measuring the cervical distensibility index in nonpregnant patients, during gestation and during the postpartum period, are presented. The ability to easily quantify the mechanical characteristics of the uterine cervix in humans throughout gestation will now allow one to carry out precise studies concerning pharmacologically induced cervical maturation or pathological modifications of the uterine cervix in cervical incompetence or threatened premature labor.