Is the beta3-adrenoceptor (ADRB3) a potential target for uterorelaxant drugs?

The management of premature birth still remains unsatisfactory. Since the relative lack of efficiency and/or safety of current tocolytic agents have been highlighted, it is necessary to develop new uterorelaxant drugs deprived of important maternal and foetal side effects. Our work reported in this review focuses on a potential new target for tocolytic drugs, the β3-adrenoceptor (ADRB3). This third type of ADRB is shown to be present and functional in human myometrium. We demonstrated that ADRB3 agonists are able to inhibit in-vitro spontaneous contractions of myometrial strips, via a cyclic AMP-mediated pathway. Furthermore, we established that ADRB3 is the predominant subtype over the ADRB2 in human myometrium and that its expression is increased in near-term myometrium, compared to non-pregnant myometrium. Finally, we reported that contrary to ADRB2, the human myometrial ADRB3 is resistant to long-term agonist-induced desensitisation. These compelling data confirm the clinical potential interest of ADRB3 agonists in the pharmacological management of preterm labour

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

Rôle des phosphodiestérases de type 4 dans la signalisation du LPS des cellules chorioniques humaines

LE KREMLIN-B.- PARIS 11-BU Méd (940432101) / SudocSudocFranceF

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-alpha and increased the release of the anti-inflammatory cytokine IL-10. PDE4 inhibition reduced cyclooxygenase-2 protein expression and PGE(2) 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

Inflammation of choriodecidua induces tumor necrosis factor alpha-mediated apoptosis of human myometrial cells.

The present study investigated the ability of human choriodecidua to induce myometrial cell apoptosis through the secretion of tumor necrosis factor alpha (TNF). The secretion of TNF was evaluated in the culture supernatants of amnion and choriodecidua explants that were exposed to the bacterial endotoxin lipopolysaccharide (LPS) to mimic inflammation. The choriodecidua explants produced more TNF than the amnion explants in response to LPS stimulation, despite the fact that the choriodecidua had lower levels of TLR4 expression. Moreover, conditioned medium obtained from LPS-treated choriodecidua explants, but not that from amnion explants, decreased the number of viable cultured myometrial cells and induced cell apoptosis by inducing the overexpression of the proapoptotic protein BAX and by decreasing the expression of the anti-apoptotic protein BCL2. Neutralization of TNF in the choriodecidua-conditioned medium reversed this effect. Exogenous TNF mimicked LPS-treated choriodecidua-conditioned medium in that it induced myometrial cell apoptosis, reduced BCL2 expression, and increased BAX expression. Using neutralizing antibodies against both subtypes of TNF receptors, we found that only TNFRSF1A participates in TNF-induced myometrial cell apoptosis. Our in vitro model of LPS-induced inflammation of human fetal membrane explants suggests a mechanism by which TNF secreted by choriodecidua governs human myometrial cell apoptosis at the end of pregnancy. These data support the hypothesis that TNF participates in the complex network of signaling processes associated with uterine involution

A role for PKCzeta in the LPS-induced translocation NF-kappaB p65 subunit in cultured myometrial cells.

Human myometrial cells respond to the endotoxin lipopolysaccharide (LPS) by activation of protein kinase C (PKC) zeta and nuclear translocation of the p65 subunit of NF-kB. Our first objective was to determine the expression of TLR4 in cultured myometrial cells. Positive immunoreactivity observed for TLR4 suggests that myometrial cells have the potential to respond to LPS. To confirm that LPS signals via TLR4, the ability of an anti-TLR4 neutralizing antibody to block LPS-induced translocation of p65 was demonstrated. To determine whether LPS-induced nuclear translocation of p65 is mediated through the PKC pathway, myometrial cells were treated with various inhibitors of the PKC isoforms already characterized in human myometrium. Neither the selective conventional PKC inhibitor nor the inhibitor of PKCdelta affected NF-kB activation. By contrast, we found that treatment of myometrial cells with an antisense against PKCzeta affect LPS-induced nuclear translocation of the p65 subunit of NF-kB. Accordingly, our data support the notion that PKCzeta is essential for LPS-induced NF-kB p65 subunit nuclear translocation in human myometrial cells

Should phosphodiesterase 5 selective inhibitors be used for uterine relaxation?

International audienceSildenafil citrate is a phosphodiesterase 5-selective inhibitor used successfully in treating erectile dysfunction. High doses of sildenafil can inhibit myometrial contractions. However, no study has demonstrated a role for phosphodiesterase 5 in myometrial contractility. No clinical trial using sildenafil to promote uterine relaxation should be initiated based on the currently available data

Anti-inflammatory and utero-relaxant effects in human myometrium of new generation phosphodiesterase 4 inhibitors.

International audienceThe anti-inflammatory and utero-relaxant effects of two potent phosphodiesterase 4 (PDE4) inhibitors of the latest generation: cilomilast (one of the most advanced PDE4 inhibitors in clinical development, reportedly more selective for PDE4D) and compound A (which displays 12-fold greater selectivity toward PDE4B and/or PDE4A than toward PDE4D) were evaluated in human uterine smooth muscle. We first established that these compounds exhibit greater efficacy in inhibiting total cAMP-PDE activity in pregnant versus nonpregnant myometrium (E(max) = 78.0% +/- 3.6% and 80.3% +/- 2.2% in pregnant versus 57% +/- 4.7% and 70.5% +/- 5.9% in nonpregnant women for compound A and cilomilast, respectively; P < 0.05 for both compounds), confirming the prominent participation of PDE4 isoforms in cAMP hydrolysis in the near-term pregnant myometrium. Using pregnant myometrial explants, we have shown that both these drugs and also rolipram, the prototype PDE4 inhibitor, produce concentration-dependent inhibition of lipopolysaccharide (LPS) induced tumor necrosis factor alpha (TNFalpha) release with similar potency in each case (pD2 = 8.0 +/- 0.5, 7.9 +/- 0.2, and 7.6 +/- 0.2 for compound A, cilomilast, and rolipram, respectively). The maximum inhibition produced is 65%. Pretreatment with forskolin or 8-bromo-cAMP mimics the PDE4 inhibitor effect. Furthermore, compound A and cilomilast both produce concentration-dependent inhibition of the spontaneous contractions of myometrial strips and are more potent in pregnant than in nonpregnant myometrium (pD2 = 7.3 +/- 0.7 and 8.1 +/- 0.3 in pregnant versus 6.2 +/- 0.9 and 6.6 +/- 0.1 in nonpregnant myometrium for compound A and cilomilast, respectively; P < 0.05 for both compounds). This demonstrates that the PDE4 isoforms involved in the mechanism of contraction are different in the pregnant and nonpregnant myometrium. Our study highlights the importance of developing PDE4 inhibitors for the pharmacological management of infection-induced preterm labor

Surfactant protein a signaling pathways in human uterine smooth muscle cells.

International audienceThe present study investigated the ability of surfactant associated protein A1 (SFTPA1), a major component of lung surfactant, to bind and serve as a signal in human cultured myometrial cells. By using ligand blot analysis with 125I-SFTPA1, we consistently identified two myometrial SFTPA1 interacting proteins (55 and 200 kDa). We found that the SFTPA1 immunoreactive protein was present in myometrial cells. We also showed by indirect immunofluorescence the nuclear translocation of RELA (also known as NFkappaB p65 subunit) after activation of myometrial cells by SFTPA1. Neutralization of TLR4 did not reverse this effect. Moreover, SFTPA1 rapidly activated mitogen-activated protein kinase 1/3 (MAPK1/3) and protein kinase C zeta (PRKCZ). The prolonged treatment of myometrial cells with SFTPA1 upregulated PTGS2 (COX2) protein levels. We next evaluated whether SFTPA1 affected the actin dynamic. Stimulation of myometrial cells with SFTPA1 markedly enhanced the intensity of the filamentous-actin pool stained with fluorescein isothiocyanate-phalloidin. Inhibition of PRKC or Rho-associated, coiled-coil containing protein kinase 1 (ROCK) reduced the SFTPA1-mediated stress fiber formation. Our data support the hypothesis that human myometrial cells express functional SFTPA1 binding sites and respond to SFTPA1 to initiate activation of signaling events related to human parturition