Macrophage-induced reactive oxygen species promote myometrial contraction and labor-associated mechanisms†.

At labor, the myometrium is infiltrated by a massive influx of macrophages that secrete high levels of pro-inflammatory cytokines inducing the expression of specific labor-associated markers. However, the interactions between myocytes and macrophages and the role of macrophages in the myometrium at labor remain to be elucidated. In this work, we studied the role of myometrium-infiltrated macrophages and their interaction with myocytes in lipopolysaccharide-induced preterm labor. A co-culture model of human primary myometrial cells and macrophages was developed and validated. Collagen lattices were used to evaluate myocyte contraction. Differentiation steps were assessed by (i) phalloidin and vinculin staining for cytoskeleton reorganization, (ii) gap junction protein alpha 1 expression and scrape loading/dye transfer with Lucifer Yellow for gap junction intercellular communication, and (iii) calcium imaging for cell excitability. We demonstrated that macrophages favored lipopolysaccharide-induced contraction and early differentiation of myometrial cells. Transwell assays showed that previous activation of macrophages by lipopolysaccharide was essential for this differentiation and that macrophage/myocyte interactions involved macrophage release of reactive oxygen species (ROS). The effects of macrophage-released ROS in myometrial cell transactivation were mimicked by H2O2, suggesting that superoxide anion is a major intermediate messenger in macrophage/myocyte crosstalk during labor. These novel findings provide the foundation for innovative approaches to managing preterm labor, specifically the use of antioxidants to inhibit the initial stages of labor before the contractile phenotype has been acquired. In addition, the co-culture model developed by our team could be used in future research to decipher pathophysiological signaling pathways or screen/develop new tocolytics.

Beta3 adrenergic receptor stimulation in human macrophages inhibits NADPHoxidase activity and induces catalase expression via PPARγ activation.

The beta3 adrenergic receptor (ß3-AR) stimulation plays a protective role against preterm labor by blocking myometrial contraction, cytokine production, remodeling and apoptosis. We previously demonstrated that macrophage-induced ROS production in the myometrium was a key element leading to the induction of all these labor-associated features. We thus aimed to investigate if the ß3-AR could be expressed in human macrophages and could trigger its protective role in the myometrium by directly inhibiting ROS production. Using lipopolysaccharide (LPS)-stimulated myometrial samples and cell co-culture experiments, we demonstrated that ß3-AR stimulation inhibits the activation of the NADPH oxidase, leading to the subsequent inhibition of ROS production by macrophages. This antioxidant effect was associated with a potent anti-inflammatory response in macrophages. Furthermore, we observed that ß3-AR leads to the expression of catalase not only in macrophages but also in myometrial cells, thereby preventing the transactivation of myometrial cells by hydrogen peroxide. Pharmacological experiments allowed us to demonstrate that these effects were driven by an Erk1/2-mediated activation of the antioxidant transcription factor PPARγ. These results suggest that ß3-AR protective effects in the myometrium could be due to its dual antioxidant properties. Further, the effects observed in a macrophage could highlight new applications in chronic inflammatory diseases.

Intra-arterial idarubicin_lipiodol without embolisation in hepatocellular carcinoma: The LIDA-B phase I trial.

BACKGROUND & AIMS: Idarubicin shows high cytotoxicity against hepatocellular carcinoma (HCC) cells, a high hepatic extraction ratio, and high lipophilicity leading to stable emulsions with lipiodol. A dose-escalation phase I trial of idarubicin_lipiodol (without embolisation) was conducted in patients with cirrhotic HCC to estimate the maximum-tolerated dose (MTD) and to assess the safety, efficacy, and pharmacokinetics of the drug, and the health-related quality of life achieved by patients. METHODS: Patients underwent two sessions of treatment with a transarterial idarubicin_lipiodol emulsion without embolisation. The idarubicin dose was escalated according to a modified continuous reassessment method. The MTD was defined as the dose closest to that causing dose-limiting toxicity (DLT) in 20% of patients. RESULTS: A group of 15 patients were enrolled, including one patient at 10 mg, four patients at 15 mg, seven patients at 20 mg, and three patients at 25 mg. Only two patients experienced DLT: oedematous ascitic decompensation and abdominal pain at 20 and 25 mg, respectively. The calculated MTD of idarubicin was 20 mg. The most frequent grade ≥3 adverse events were biological. One month after the second session, the objective response rate was 29% (complete response, 0%; partial response, 29%) based on modified Response Evaluation Criteria In Solid Tumours. The median time to progression was 5.4 months [95% confidence limit (CI) 3.0-14.6 months] and median overall survival was 20.6 months (95% CI 5.7-28.7 months). Pharmacokinetic analysis of idarubicin showed that the mean Cmax of idarubicin after intra-arterial injection of the idarubicin-lipiodol emulsion is approximately half the Cmax after intravenous administration. Health-related quality of life results confirmed the good safety results associated with use of the drug. CONCLUSIONS: The MTD of idarubicin was 20 mg after two chemolipiodolisation sessions. Encouraging safety results, and patient responses and survival were observed. A phase II trial has been scheduled. LAY SUMMARY: There is a need for transarterial regimens that improve the responses and survival of patients with unresectable HCC. In this phase I trial, we showed that two sessions of treatment with a transarterial idarubicin_lipiodol emulsion without embolisation was well tolerated and gave promising efficacy in terms of tumour control and patient survival.

Glutathione prevents preterm parturition and fetal death by targeting macrophage-induced reactive oxygen species production in the myometrium.

Preterm birth is an inflammatory process resulting from the massive infiltration of innate immune cells and the production of proinflammatory cytokines in the myometrium. However, proinflammatory cytokines, which induce labor in vivo, fail to induce labor-associated features in human myometrial cells (MCs). We thus aimed to investigate if reactive oxygen species (ROS) production could be the missing step between immune cell activation and MC response. Indeed, we found that ROS production is increased in the human preterm laboring myometrium (27% ROS producing cells, respectively, versus 2% in nonlaboring controls), with 90% ROS production in macrophages. Using LPS-stimulated myometrial samples and cell coculture experiments, we demonstrated that ROS production is required for labor onset. Furthermore, we showed that ROS are required first in the NADPH oxidase (NADPHox)-2/NF-κB-dependent macrophage response to inflammatory stimuli but, more importantly, to trigger macrophage-induced MCs transactivation. Remarkably, in a murine model of LPS-induced preterm labor (inducing delivery within 17 hours, with no pup survival), cotreatment with glutathione delayed labor onset up to 94 hours and prevented in utero fetal distress, allowing 46% pups to survive. These results suggest that targeting ROS production with the macrophage-permeable antioxidant glutathione could constitute a promising strategy to prevent preterm birth.

Improved stability of lipiodol-drug emulsion for transarterial chemoembolisation of hepatocellular carcinoma results in improved pharmacokinetic profile: Proof of concept using idarubicin.

OBJECTIVES: To investigate the relationship between the improved stability of an anticancer drug-lipiodol emulsion and pharmacokinetic (PK) profile for transarterial chemoembolisation (TACE) of hepatocellular carcinoma (HCC). METHODS: The stability of four doxorubicin- or idarubicin-lipiodol emulsions was evaluated over 7 days. PK and clinical data were recorded after TACE with the most stable emulsion in eight unresectable HCC patients, after institutional review board approval. RESULTS: The most stable emulsion was the one that combined idarubicin and lipiodol (1:2 v:v). At 7 days, the percentages of aqueous, persisting emulsion and oily phases were 50-0-50, 33-0-67, 31-39-30, and 10-90-0 for the doxorubicin-lipiodol (1:1 v:v), doxorubicin-lipiodol (1:2 v:v), idarubicin-lipiodol (1:1 v:v), and the idarubicin-lipiodol (1:2 v:v) emulsion, respectively. After TACE, mean idarubicin Cmax and AUC0-24h were 12.5 ± 9.4 ng/mL and 52 ± 16 ng/mL*h. Within 24 h after injection, 40% of the idarubicin was in the liver, either in vessels, tumours, or hepatocytes. During the 2 months after TACE, no clinical grade >3 adverse events occurred. One complete response, five partial responses, one stabilisation, and one progression were observed at 2 months. CONCLUSION: This study showed a promising and favourable PK and safety profile for the idarubicin-lipiodol (1:2 v:v) emulsion for TACE. KEY POINTS: • Transarterial chemoembolisation (TACE) regimens that improve survival in hepatocellular carcinoma are needed. • Improved emulsion stability for TACE resulted in a favourable pharmacokinetic profile. • Preliminary safety and efficacy data for the idarubicin-lipiodol emulsion for TACE were encouraging.

Idarubicin-loaded ONCOZENE drug-eluting embolic agents for chemoembolization of hepatocellular carcinoma: in vitro loading and release and in vivo pharmacokinetics.

PURPOSE: To present in vitro loading and release characteristics of idarubicin with ONCOZENE (CeloNova BioSciences, Inc, San Antonio, Texas) drug-eluting embolic (DEE) agents and in vivo pharmacokinetics data after transarterial chemoembolization with idarubicin-loaded ONCOZENE DEE agents in patients with hepatocellular carcinoma. MATERIALS AND METHODS: Loading efficacy of idarubicin with ONCOZENE DEE agents 100 µm and DC Bead (Biocompatibles UK Ltd, Farnham, United Kingdom) DEE agents 100-300 µm was monitored at 10, 20, and 30 minutes loading time by high-pressure liquid chromatography. A T-apparatus was used to monitor the release of idarubicin from the two types of DEE agents over 12 hours. Clinical and 24-hour pharmacokinetics data were recorded after transarterial chemoembolization with idarubicin-loaded ONCOZENE DEE agents in four patients with unresectable hepatocellular carcinoma. RESULTS: Idarubicin loading in ONCOZENE DEE agents was > 99% at 10 minutes. Time to reach 75% of the release plateau level was 37 minutes ± 6 for DC Bead DEE agents and 170 minutes ± 19 for ONCOZENE DEE agents both loaded with idarubicin 10 mg/mL. After transarterial chemoembolization with idarubicin-loaded ONCOZENE DEE agents, three partial responses and one complete response were observed with only two asymptomatic grade 3 biologic adverse events. Median time to maximum concentration for idarubicin in patients was 10 minutes, and mean maximum concentration was 4.9 µg/L ± 1.7. Mean area under the concentration-time curve from 0-24 hours was equal to 29.5 µg.h/L ± 20.5. CONCLUSIONS: ONCOZENE DEE agents show promising results with very fast loading ability, a favorable in vivo pharmacokinetics profile with a sustained release of idarubicin during the first 24 hours, and encouraging safety and responses. Histopathologic and clinical studies are needed to evaluate idarubicin release around the DEE agents in tumor tissue and to confirm safety and efficacy.

Biphasic Erk1/2 activation sequentially involving Gs and Gi signaling is required in beta3-adrenergic receptor-induced primary smooth muscle cell proliferation.

The beta3 adrenergic receptor (B3-AR) reportedly induces cell proliferation, but the signaling pathways that were proposed, involving either Gs or Gi coupling, remain controversial. To further investigate the role of G protein coupling in B3-AR induced proliferation, we stimulated primary human myometrial smooth muscle cells with SAR150640 (B3-AR agonist) in the absence or presence of variable G-protein inhibitors. Specific B3-AR stimulation led to an Erk1/2 induced proliferation. We observed that the proliferative effects of B3-AR require two Erk1/2 activation peaks (the first after 3min, the second at 8h). Erk1/2 activation at 3min was mimicked by forskolin (adenylyl-cyclase activator), and was resistant to pertussis toxin (Gi inhibitor), suggesting a Gs protein signaling. This first signaling also required the downstream Gs signaling effectors PKA and Src. However, Erk1/2 activation at 8h turned out to be pertussis toxin-dependent, and PKA-independent, indicating a Gi signaling pathway in which Src and PI3K were required. The pharmacological inhibition of both the Gs and Gi pathway abolished B3-AR-induced proliferation. Altogether, these data indicate that B3-AR-induced proliferation depends on the biphasic activation of Erk1/2 sequentially induced by the Gs/PKA/Src and Gi/Src/PI3K signaling pathways.

Systemic increase in human maternal circulating CD14+CD16- MCP-1+ monocytes as a marker of labor.

OBJECTIVES: To study the influence of pregnancy and labor on the proportion and level of activation of monocyte subpopulations in human pregnancy. STUDY DESIGN: Peripheral blood samples were obtained from healthy nonpregnant women (n = 6); women in the third-trimester of healthy pregnancies (n = 18) and women with preterm premature rupture of membranes (n = 46), just before delivery for the last 2 groups. Monocyte subpopulations were characterized by flow cytometry using CD14, CD16, and activation level using macrophage chemoattractant protein-1 (MCP-1) and CCR2 antibodies. RESULTS: The relative proportion of each monocyte subset in nonpregnant women was similar to that in women with healthy or complicated pregnancies. However, pregnancy was associated with a significant decrease in MCP-1 expressing monocytes (79.5% ± 19.8% vs 9.3% ± 6.8% and 11.9% ± 8.3% for nonpregnant, healthy pregnancy, and preterm premature rupture of membranes (respectively, P < .05). Spontaneous labor was associated with a return to nonpregnant values for the proportion of MCP-1 expressing monocytes in both normal (74.4% ± 16.9) and preterm premature rupture of membranes pregnancy (68.4% ± 35.6), irrespective of the mode of delivery (vaginal or cesarean section). This was not observed in women who delivered without spontaneous labor onset. CCR-2 (MCP-1 receptor) expression was not modified in monocytes at the time of labor, but was significantly increased in granulocytes (3646 ± 1080 vs 7338 ± 2718 for nonlaboring and laboring preterm premature rupture of membranes, respectively, P < .05) CONCLUSION: In light of previous reports of a role for MCP-1 in labor, our results suggest the downregulation of activation levels of monocytes, via MCP-1 expression might be involved in maternofetal immune tolerance. Monocyte reactivation might be associated with labor.

Effects of leptin on lipopolysaccharide-induced remodeling in an in vitro model of human myometrial inflammation.

Reorganization of myometrial extracellular matrix (ECM) is essential for the uterus to achieve powerful synchronous contractions during labor. Remodeling of the ECM has been implicated in membrane rupture and cervical ripening. Because maternal obesity is associated with both delivery disorders and elevated circulating leptin levels, this study aimed to assess the ability of leptin to interfere with lipopolysaccharide (LPS)-induced myometrial ECM remodeling. Myometrial biopsy samples were obtained from women undergoing cesarean delivery before labor onset. Myometrial explants were incubated for 48 h with LPS and leptin. LPS challenge was associated with a marked decrease in collagen content and in heat shock protein (HSP) 47 expression, reflecting a disruption in collagen synthesis and an increase in matrix metalloproteinase (MMP) 2 and MMP9 activity and in MMP2, MMP9, and MMP13 expression. Leptin prevented an LPS-induced decrease in myometrial collagen content in a concentration-dependent manner. This effect was associated with an increase in HSP47 expression and a decrease in MMP2 and MMP9 activity and expression. These results show that leptin prevents LPS-induced myometrial remodeling through collagen synthesis stimulation and inhibition of MMP2 and MMP9. Our study strengthens the hypothesis that leptin plays a role in the development of obesity-related delivery disorders.

Wound Healing versus Metastasis: Role of Oxidative Stress.

Many signaling pathways, molecular and cellular actors which are critical for wound healing have been implicated in cancer metastasis. These two conditions are a complex succession of cellular biological events and accurate regulation of these events is essential. Apart from inflammation, macrophages-released ROS arise as major regulators of these processes. But, whatever the pathology concerned, oxidative stress is a complicated phenomenon to control and requires a finely tuned balance over the different stages and responding cells. This review provides an overview of the pivotal role of oxidative stress in both wound healing and metastasis, encompassing the contribution of macrophages. Indeed, macrophages are major ROS producers but also appear as their targets since ROS interfere with their differentiation and function. Elucidating ROS functions in wound healing and metastatic spread may allow the development of innovative therapeutic strategies involving redox modulators.

Leptin-Induced HLA-G Inhibits Myometrial Contraction and Differentiation.

Maternal obesity is associated with a wide spectrum of labour disorders, including preterm birth. Leptin, a pro-inflammatory adipokine and a key factor of obesity, is suspected to play a major role in these disorders. OB-R, its receptor, is expressed on macrophages and myocytes, two cell types critical for labour onset. Macrophages secrete reactive oxygen species/pro-inflammatory cytokines, responsible for myometrial differentiation while myocytes control uterine contractions. In this study, we assessed the effect of leptin on myometrial contraction and differentiation using our validated co-culture model of human primary macrophages and myocytes. We demonstrated that leptin had a different effect on myocytes and macrophages depending on the dose. A low leptin concentration induced a tocolytic effect by preventing myocytes' contraction, differentiation, and macrophage-induced ROS production. Additionally, leptin led to an increase in HLA-G expression, suggesting that the tocolytic effect of leptin may be driven by HLA-G, a tolerogenic molecule. Finally, we observed that recombinant HLA-G also prevented LPS-induced ROS production by macrophages. Altogether, these data provide a putative molecular mechanism by which leptin may induce immune tolerance and therefore interfere with labour-associated mechanisms. Therefore, HLA-G represents a potential innovative therapeutic target in the pharmacological management of preterm labour.

Effects of leptin on lipopolysaccharide-induced myometrial apoptosis in an in vitro human model of chorioamnionitis.

OBJECTIVE: This study was aimed at assessing the role of leptin on human myometrium, by studying its receptor expression in pregnant myometrium and the interaction of leptin with inflammation-induced apoptosis. STUDY DESIGN: Myometrial samples were obtained from women with uncomplicated pregnancies who underwent cesarean delivery at term before labor onset. The effect of leptin on apoptosis was assessed by the incubation of myometrial strips with leptin (10(-10) to 10(-8) mol/L; 48 hours) before lipopolysaccharide treatment (10 µg/mL; 48 hours). RESULTS: Long and short leptin receptor isoforms were expressed in myometrial cells of pregnant women. Leptin prevented lipopolysaccharide-induced apoptosis, in a concentration-dependent manner, by down-regulating cleaved caspase-3 and BCL2-associated X protein and up-regulating BCL2 expression. This effect was mediated specifically through leptin receptor stimulation, followed by ERK1/2 signaling pathway activation. CONCLUSION: These results suggest a new potential pathway that is involved in delivery disorders of obese women and propose a role for the leptin-induced inhibition of myometrial apoptosis in the development of such disorders.

Lipoproteins LDL versus HDL as nanocarriers to target either cancer cells or macrophages.

In this work, we have explored natural unmodified low- and high-density lipoproteins (LDL and HDL, respectively) as selective delivery vectors in colorectal cancer therapy. We show in vitro in cultured cells and in vivo (NanoSPECT/CT) in the CT-26 mice colorectal cancer model that LDLs are mainly taken up by cancer cells, while HDLs are preferentially taken up by macrophages. We loaded LDLs with cisplatin and HDLs with the heat shock protein-70 inhibitor AC1LINNC, turning them into a pair of "Trojan horses" delivering drugs selectively to their target cells as demonstrated in vitro in human colorectal cancer cells and macrophages, and in vivo. Coupling of the drugs to lipoproteins and stability was assessed by mass spectometry and raman spectrometry analysis. Cisplatin vectorized in LDLs led to better tumor growth suppression with strongly reduced adverse effects such as renal or liver toxicity. AC1LINNC vectorized into HDLs induced a strong oxidative burst in macrophages and innate anticancer immune response. Cumulative antitumor effect was observed for both drug-loaded lipoproteins. Altogether, our data show that lipoproteins from patient blood can be used as natural nanocarriers allowing cell-specific targeting, paving the way toward more efficient, safer, and personalized use of chemotherapeutic and immunotherapeutic drugs in cancer.

[Accreditation of the toxicological screening: recommendations of the SFBC-SFTA group]. / Accréditation du criblage toxicologique : recommandations du groupe SFBC-SFTA.

Toxicological screening is a specific approach to analytical toxicology that uses analytical tools such as GC-MS, LC-UV (diode array) or LC-MS. Toxicological screening allows the detection and simultaneous identification of a large number of compounds. The results may be based on the use of one or more techniques. As part of the accreditation process for medical biology examinations according to standard NF EN ISO 15189, the group from SFTA and SFBC recommends an approach to accredit toxicological screening. Indeed, the complexity of the accreditation of this analysis comes in particular from the high number of compounds that can be detected. Validation parameters are discussed in the specific context of toxicological screening by considering two distinct approaches: the simple identification of compounds, or the identification and estimation of a range of concentration related to clinical outcomes.

Dose-dependent biphasic leptin-induced proliferation is caused by non-specific IL-6/NF-κB pathway activation in human myometrial cells.

BACKGROUND AND PURPOSE: Leptin, an adipokine synthesized by the placenta during pregnancy, has been proposed for the management of preterm labour (PTL), as it is able to prevent in vitro uterine contractility and remodelling associated with labour onset. Another common feature of labour onset is the phenotypic switch of myometrial smooth muscle cells from a proliferative to a hypertrophic state. As proliferative effects have been demonstrated for leptin in other tissues, we aimed to investigate its ability to induce myometrial proliferation and thus to maintain uterine quiescence. EXPERIMENTAL APPROACH: We stimulated human primary myometrial smooth muscle cells with leptin in the presence or absence of receptor antagonists or signalling pathway inhibitors. KEY RESULTS: Leptin induced myometrial cell proliferation in a biphasic manner. At 6.25 ng · mL(-1), leptin-induced proliferation was mediated by the leptin receptor and required the early activation of ERK1/2. At a concentration above 25 ng · mL(-1), leptin induced direct non-specific stimulation of the IL-6 receptor, leading to NF-κB activation, and exerted anti-proliferative effects. However, at 50 ng · mL(-1), leptin re-induces proliferation via IL-6 receptor stimulation that requires STAT3 and delayed ERK1/2 activation. CONCLUSIONS AND IMPLICATIONS: These data bring new insights into leptin signalling-induced myometrial proliferation and its interrelationship with the IL-6/IL-6 receptor axis. In the light of our previous work, the present study emphasizes the potential value of leptin in the pharmacological management of PTL and it also strengthens the hypothesis that leptin might be a contributory factor in the parturition-related disorders observed in obese women.