SAR150640, a selective beta3-adrenoceptor agonist, prevents human myometrial remodelling and activation of matrix metalloproteinase in an in vitro model of chorioamnionitis

Abstract

Background and purpose: The uterine pathophysiology underlying inflammatory conditions such as chorioamnionitis remains largely unclear. As we have shown that beta(3)-adrenoceptors act as regulators of myometrial inflammation, we wanted to investigate the potential role of beta(3)-adrenoceptors in preventing uterine remodelling induced by inflammation.

Experimental approach: The consequences of human chorioamnionitis on myometrial remodelling were characterized by Sirius Red staining and metalloproteinase (MMP) expression, and compared with the effects of incubating human myometrial samples with Escherichia coli lipopolysaccharide (LPS) in vitro. We also assessed the effect of SAR150640, a selective beta(3)-adrenoceptor agonist, on the production and activity of MMPs.

Key results: Chorioamnionitis was associated with a 46% decrease in total collagen, as well as over-expression of MMP2 (+61%) and MMP9 (+84%); both effects were reproduced by incubation with LPS (10 microg x mL(-1), 48 h). LPS-induced over-expression of MMP2 and MMP9 in normal human myometrium was paralleled by an overactivity of the proteins. Both over-expression and overactivity were prevented by the beta(3)-adrenoceptor agonist SAR150640 in a concentration-dependent manner. SAR150640, by itself, did not exhibit any effect on MMP production in control tissues.

Conclusions and implications: This study shows that inflammation was associated with an intense remodelling of human myometrium, a process likely to be explained by MMP activation. Our study emphasizes the potential therapeutic relevance of beta(3)-adrenoceptor agonists to the treatment of preterm labour and other uterine inflammatory conditions.

Figure 1

Time-course of the effect of LPS (10 µg·mL⁻¹) on the levels of the transcripts for MMP1, MMP2, MMP3 and MMP9, and on the activities of pro-MMP9 and pro-MMP2. Myometrial explants were incubated with LPS (10 µg·mL⁻¹) for 6–72 h. MMP transcripts were assessed using quantitative real-time RT-PCR, and proteolytic activity of pro-MMP-9 and pro-MMP2 secreted into the culture medium was assessed by gelatin zymography. Experiments were performed in triplicate, respectively, on three myometrial samples for RT-PCR and on two supernatants for zymography analysis, each obtained from a different woman. A 72 h time-matched control without LPS stimulation was performed (72 h-C). *P < 0.05, **P < 0.01 versus control 3 h.

Figure 3

Chorioamnionitis and LPS stimulation (10 µg·mL⁻¹, 48 h) are associated with MMP9 and MMP2 over-expression. Western blot experiments performed on myometrial samples obtained from women with uncomplicated pregnancies, stimulated or not with LPS, and from women with confirmed chorioamnionitis, showing MMP9 (A) and MMP2 (B) expression. n= 3 for chorioamionitis and 5 for LPS experiment, which were all performed in duplicate. Controls, control time-matched experiments; LPS, 10 µg·mL⁻¹ LPS 48 h; CA, chorioamnionitis. **P < 0.01 and ***P < 0.001 versus controls.

Figure 2

Chorioamnionitis and LPS stimulation (10 µg·mL⁻¹, 48 h) are associated with diminution of total collagen. Sirius Red staining of human myometrial sections (×100 magnification). (A–F) Negative control, chorioamnionitis, LPS stimulation (10 µg·mL⁻¹, 48 h), without and with SAR 150640 at 0.1, 1 and 10 µM respectively. (G) Semi-quantification of total collagen, expressed in % of fluorescence mm⁻². CTRL, control; LPS, 10 µg·mL⁻¹ LPS, 48 h; CA, chorioamnionitis. Experiments were performed in myometrial samples obtained from three different women with chorioamnionitis (CA group) and in six myometrial samples obtained from women with uncomplicated pregnancies (n= 3) and stimulated with LPS (n= 3), without and with SAR 150640 at 0.1, 1 and 10 µM. ***P < 0.001 versus control and ###P < 0.001 versus chorioamnionitis or LPS stimulation.

Figure 4

SAR150640 prevents LPS-induced MMP9 activity and expression. Gelatin zymography analysis (A) with semi-quantification of pro-MMP9 activity (B) and Western blot analysis (C) with semi-quantification (D) showing the expression of MMP9 protein. Legends: CTRL, control time-matched experiments; LPS, 10 µg·mL⁻¹ LPS 48 h; SAR150640 (0.1, 1, 10 µM) was added 20 min prior to LPS stimulation. Experiments were performed, respectively, on four and seven myometrial samples for zymography and Western blot analysis, each obtained from a different woman. ###P < 0.001 versus controls, ***P < 0.001 versus LPS.

Figure 5

SAR150640 prevents LPS-induced MMP2 activity and expression. Gelatin zymography analysis (A) with semi-quantification of pro-MMP2/MMP2 activity (B) and Western blot analysis (C) with semi-quantification (D) showing the expression of MMP2 protein. Legends: CTRL, control time-matched experiments; LPS, 10 µg·mL⁻¹ LPS 48 h; SAR150640 (0.1, 1, 10 µM) was added 20 min prior to LPS stimulation. Experiments were performed, respectively, on four and six myometrial samples for zymography and Western blot analysis, each obtained from a different woman. ###P < 0.001 versus controls; *P < 0.05, **P < 0.01 and ***P < 0.0001 versus LPS.

Figure 6

The effect of SAR150640 on LPS-induced MMP9 expression is mediated only by β₃-adrenoceptors. Western blot analysis (A) with semi-quantification (B) showing the expression of MMP9 protein. Controls, time-matched experiments; LPS,10 µg·mL⁻¹ LPS 48 h. SAR150640 (0.1, 1, 10 µM) was added 20 min prior to LPS stimulation. Experiments were performed on six myometrial samples, each obtained from a different woman. ***P < 0.001 versus controls.