Tocolytic effect of a Rho-kinase inhibitor in a mouse model of lipopolysaccharide-induced preterm delivery

doi:10.1016/j.ajog.2004.09.016

American Journal of Obstetrics and Gynecology

Volume 192, Issue 3, March 2005, Pages 903-908

https://doi.org/10.1016/j.ajog.2004.09.016 Get rights and content

Objective

The small guanosine triphosphatase RhoA/Rho-kinase cascade has been implicated in uterine contraction. Our purpose was to evaluate the tocolytic effect of a Rho-kinase inhibitor, Y-27632, in lipopolysaccharide-induced preterm delivery in mice.

Study design

We used an animal model of lipopolysaccharide-induced preterm delivery in C3H/HeN × B6D2F1 pregnant mice. Y-27632 was delivered continuously through an osmotic pump that was implanted into the peritoneal cavity 6 hours before lipopolysaccharide treatment. The primary outcome was the preterm delivery rate. To further study the possible involvement of this cascade in lipopolysaccharide-induced preterm delivery, we determined the effect of lipopolysaccharide and prostaglandin F_2α on RhoA activation in mouse myometrial cells and uterine smooth muscle tissues.

Results

The rate of preterm delivery for lipopolysaccharide-treated animals was 94.4%. The administration of Y-27632 (1 or 10 mg/kg/d) significantly reduced the preterm delivery rate to 61.1% or 15.8%, respectively. The level of guanosine triphosphate–bound RhoA was increased after the addition of lipopolysaccharide or prostaglandin F₂α.

Conclusion

The RhoA/Rho-kinase cascade is involved in lipopolysaccharide-induced preterm delivery, which suggests that Rho-kinase could be used as a new therapeutic target for the prevention of preterm labor.

Section snippets

Material

C3H/HeN female mice and B6D2F1 male mice were purchased from Charles River Japan (Yokohama, Japan). Y-27632, a specific inhibitor of Rho-kinase, was provided by Mitsubishi WelFide Corporation (Osaka, Japan). Y-27632 was dissolved in distilled water as a stock solution (10 mmol/L) and stored at −20°C until used. Osmotic pumps (model 1007D) were obtained from ALZET Osmotic Pumps (Cupertino, Calif). Lipopolysaccharide (Escherichia coli, serotype 055:B5), trypsin, DNase, and prostaglandin F_2α were

Effect of Y-27632 on lipopolysaccharide-induced preterm delivery

We evaluated the Rho-kinase inhibitor Y-27632 for its tocolytic effects on mice and used a lipopolysaccharide-induced preterm delivery mouse model. In group I, all pregnant C3H/HeN × B6D2F1 mice were delivered of pups at term at day 19 or 20 (Table). In group II, C3H/HeN × B6D2F1 pregnant mice had a high incidence (94.4%) of preterm delivery (Table). Delivery occurred without any maternal death in all cases. All preterm deliveries at <16 gestational days resulted in stillbirths, and no live

Comment

In myometrium, as in other smooth muscles, an increase in free Ca²⁺ concentration is of great importance in the regulation of contractions.²⁰ Calcium ions within smooth muscle cells bind with the Ca²⁺-binding protein calmodulin and activate myosin light-chain kinase.¹⁹ Prostaglandin and oxytocin are known to stimulate uterine contractions by facilitating cytoplasmatic influx of calcium through Ca²⁺ channels and intracellular Ca²⁺ mobilization.²⁰ Therefore, agents such as Ca²⁺ channel blockers

Acknowledgments

The Y-27632 was provided by WelFide Corporation, Osaka, Japan.

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It was found that pro-inflammatory and vasoconstrictor PGF2α level is increased in DHEA-treated mice [44]. In addition, PGF2α has been shown to induce calcium sensitization in both human [45] and mouse [46] myometria. In our study, although both Y-27632 and fasudil inhibited the PGF2α induced contractions, this suppressive effect was more effectively observed by Y-27632.
The aim of this study was to investigate the expression of RhoA/Rho-kinase in the uterus and the effect of Rho-kinase inhibitors on uterine contractions of dehydroepiandrosterone (DHEA) induced polycystic ovary syndrome (PCOS) rats. Forty-four female Sprague-Dawley (21 days old) rats divided into three groups: The control group (n = 14, any procedure was not performed), vehicle group (n = 14, 0.2 ml of sesame oil, subcutaneous injection, 20 days) and PCOS group (n = 16, DHEA 6 mg/100 g in 0.2 ml of sesame oil, subcutaneous injection, 20 days). The myometrium thickness and uterine wet weight were assessed. The mRNA and protein expressions of Rho A, the effect of Rho-kinase inhibitors (fasudil and Y-27632) on KCl, carbachol, and PGF2α induced contractions were evaluated in the uterus. In the PCOS group, the myometrium thickness and uterine wet weight significantly increased compared to the control group and vehicle group. The mRNA expression level and the immunoreactive score of Rho A, ROCK 1, ROCK 2 were similar in all groups. In the PCOS group, KCl, carbachol, and PGF2α induced uterine contractions significantly increased compared to the control group and vehicle group. Fasudil and Y-27632 significantly inhibited KCl, carbachol, and PGF2α induced uterine contractions in all groups. In conclusion, the expression of Rho A, ROCK 1, ROCK 2 not changed although myometrium thickness, uterine wet weight and the contractile responses of uterus increased in the PCOS group. The results suggest that the Rho-kinase inhibitors effectively suppressed increased contractions in the PCOS group they might be potential therapeutic agents.
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Because IMD-0560 successfully inhibited IL-6 production from hAMSCs via the inhibition of IKK activation, we asked whether anticytokine therapy using this drug can prevent PTD in an LPS-induced PTD mouse model. C3H/HeN female mice were pair-mated with B6D2F1 male mice to deliver preterm in response to low-dose LPS (double doses of 50 μg/kg at 3-hour intervals), as we and others have previously reported.15,30,32 LPS administration induced PTD reproducibly [12 of 12 (100%)] without any apparent adverse effects on the pregnant mice.
Preterm delivery (PTD) remains a serious challenge in perinatology. Intrauterine infection and/or inflammation, followed by increased inflammatory cytokines, represented by IL-6, are involved in this pathology. Our aim was to identify IL-6–producing cells in the placenta and to analyze the potential of targeting IκB kinase β (IKKβ) signaling to suppress IL-6 production for the treatment of PTD. Immunohistochemical analyses using placentas complicated with severe chorioamnionitis revealed that IL-6 is mainly expressed in human amniotic mesenchymal stromal cells (hAMSCs). Primary hAMSCs were collected, and strong IL-6 expression was confirmed. In hAMSCs, the treatment of tumor necrosis factor-α or IL-1β drastically induced IL-6 production, followed by the phosphorylation of IKKs. A novel IKKβ inhibitor, IMD-0560, almost completely inhibited IL-6 production from hAMSCs. Using an experimental lipopolysaccharide-induced PTD mouse model, the therapeutic potential of IMD-0560 was examined. IMD-0560 was delivered vaginally 4 hours before lipopolysaccharide administration. Mice in the IMD-0560 (30 mg/kg, twice a day) group had a significantly lower rate of PTD [10 of 22 (45%)] without any apparent adverse events on the mice and their pups. In uteri collected from mice, IMD-0560 inhibited not only IL-6 production but also production of related cytokines, such as keratinocyte-derived protein chemokine/CXCL1, macrophage inflammatory protein-2/CXCL2, and monocyte chemoattractant protein-1/chemokine ligand 2. Targeting IKKβ signaling shows promising effects through the suppression of these cytokines and can be explored as a future option for the prevention of PTD.
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In this chapter we will summarize the scientific results of animal studies that form the basis of our current understanding of the molecular mechanisms regulating the process of parturition in humans. Such knowledge is critical to develop effective intervention strategies to prevent the significant problem of preterm birth (PTB). PTB is a syndrome with many etiologies and redundant pathways for which we have limited understanding. In some cases, PTB represents a premature triggering of the normal events occurring during term parturition. The collection of human-derived tissues (fetal and maternal) for studying parturition is strictly regulated by clinical and ethical considerations. Therefore, the use of animal (i.e., mouse) models of labor is important for optimal experimental sample collection before, during, and after delivery. An additional advantage of the mouse as a model for PTB is the ability to manipulate genes involved in the pathways regulating parturition, which is not possible in humans.
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The suppression of uterine contractility by ROK inhibition is in keeping with previous findings.29-33 Further, in mice, lipopolysaccharide-induced preterm delivery was delayed significantly with the ROK inhibitor Y-27632.34 However, in contrast to our results, Y-27632 was shown to have a greater effect in strips from late pregnant mice compared with nonpregnant animals.31
Endogenous uterine agonists can activate numerous signaling pathways to effect increased force. Our objective was to assess expression of key constituents of these pathways, in alliance with contractile function, through late gestation and during term and preterm labor.
Using myography, we measured the response to 3 agonists compared with depolarization alone (K⁺, 124 mEq/L) and calculated agonist/depolarization ratio. We measured gene expression using quantitative reverse transcription–polymerase chain reaction.
Contractile responsiveness to depolarization alone, oxytocin, or endothelin-1 increased during pregnancy compared with nonpregnant animals. The agonist/depolarization ratio did not change during uterine activation or parturition. Inhibition of rhoA-associated kinase decreased responses to oxytocin in all tissues, but significantly more during uterine activation. Expression of rhoA and rhoA-associated kinase was increased significantly in active labor at term or preterm.
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Our findings underscore the importance of FP receptor signaling through the Gα12-Rho-ROCK pathway as a pharmacological target in the management of parturition and preterm labor. Our in vitro and in vivo data are consistent with both the observations that RhoA activity is increased in the myometrium during pregnancy, and that inhibition of ROCK blocks both PGF2α- and LPS-induced preterm labor in mice (48–50). However, the extent to which MAPK contributes to myometrial contraction and preterm labor remains an open question (51).
The prostaglandin F2α (PGF2α) receptor (FP) is a key regulator of parturition and a target for pharmacological management of preterm labor. However, an incomplete understanding of signaling pathways regulating myometrial contraction hinders the development of improved therapeutics. Here we used a peptidomimetic inhibitor of parturition in mice, PDC113.824, whose structure was based on the NH₂-terminal region of the second extracellular loop of FP receptor, to gain mechanistic insight underlying FP receptor-mediated cell responses in the context of parturition. We show that PDC113.824 not only delayed normal parturition in mice but also that it inhibited both PGF2α- and lipopolysaccharide-induced preterm labor. PDC113.824 inhibited PGF2α-mediated, Gα₁₂-dependent activation of the Rho/ROCK signaling pathways, actin remodeling, and contraction of human myometrial cells likely by acting as a non-competitive, allosteric modulator of PGF2α binding. In contrast to its negative allosteric modulating effects on Rho/ROCK signaling, PDC113.824 acted as a positive allosteric modulator on PGF2α-mediated protein kinase C and ERK1/2 signaling. This bias in receptor-dependent signaling was explained by an increase in FP receptor coupling to Gα_q, at the expense of coupling to Gα₁₂. Our findings regarding the allosteric and biased nature of PDC113.824 offer new mechanistic insights into FP receptor signaling relevant to parturition and suggest novel therapeutic opportunities for the development of new tocolytic drugs.
Regulation of the uterine contractile apparatus and cytoskeleton
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Parturition at term, the end stage of a successful pregnancy, occurs as a result of powerful, co-ordinated and periodic contractions of uterine smooth muscle (myometrium). To occur in a propitious manner, a high degree of control over the activation of a myometrial cell is required. We review the molecular mechanisms and structural composition of myometrial cells that may contribute to their increased contractile capacity at term. We focus attention on pathways that lead to the activation of filamentous networks traditionally labeled ‘contractile’ or ‘cytoskeletal’ yet draw attention to the fact that functional discrimination between these systems is not absolute.

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: Supported in part by Grants-in-Aid for Scientific Research (No.14571559 to M.T. and 14370532 to K.T.) from the Japanese Ministry of Education, Culture, Sports, Science and Technology (Tokyo, Japan).

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General Obstetrics and Gynecology: ObstetricsTocolytic effect of a Rho-kinase inhibitor in a mouse model of lipopolysaccharide-induced preterm delivery

Objective

Study design

Results

Conclusion

Section snippets

Material

Effect of Y-27632 on lipopolysaccharide-induced preterm delivery

Comment

Acknowledgments

Am J Obstet Gynecol

Am J Obstet Gynecol

J Biol Chem

Biochem Biophys Res Commun

Biochem Biophys Res Commun

Am J Obstet Gynecol

Am J Obstet Gynecol

Semin Perinatol

Am J Obstet Gynecol

Am J Obstet Gynecol

Am J Obstet Gynecol

Prevention of premature birth

N Engl J Med

The role of systemic and intrauterine infection in preterm labor

The role of infection in the etiology of preterm birth

Obstet Gynecol

General Obstetrics and Gynecology: Obstetrics
Tocolytic effect of a Rho-kinase inhibitor in a mouse model of lipopolysaccharide-induced preterm delivery