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International audience; Objective: To explore the regulatory role of soluble CD146 (sCD146) and its interaction with galectin-1 (Gal1) in placenta-mediated complications of pregnancy.Design: Prospective pilot and experimental studies.Setting: University-affiliated hospital and academic research laboratory.Patient(s): One hundred fifteen women divided into three groups: 30 healthy, nonpregnant women, 50 women with normal pregnancies, and 35 with placenta-mediated pregnancy complications.Intervention(s): Wound-healing experiments were conducted to study trophoblast migration.Main outcome measure(s): Quantification of sCD146 and Gal1 by enzyme-linked immunosorbent assay. Analysis of trophoblast migration by wound closure.Result(s): Concomitant detection of sCD146 and Gal1 showed lower sCD146 and higher Gal1 concentrations in women with normal pregnancies compared with nonpregnant women. In addition, follow-up of these women revealed a decrease in sCD146 associated with an increase in Gal1 throughout pregnancy. In contrast, in women with preeclampsia, we found significantly higher sCD146 concentrations compared with women with normal pregnancies and no modification of Gal1. We emphasize the opposing effects of sCD146 and Gal, since, unlike Gal1, sCD146 inhibits trophoblast migration. Moreover, the migratory effect of Gal1 was abrogated with the use of an anti-CD146 blocking antibody or the use of small interfering RNA to silence VEGFR2 expression. This suggests that trophoblast migration is mediated though the interaction of Gal1 with CD146, further activating the VEGFR2 signaling pathway. Significantly, sCD146 blocked the migratory effects of Gal1 on trophoblasts and inhibited its secretion, suggesting that sCD146 acts as a ligand trap.Conclusion(s): Soluble CD146 could be proposed as a biomarker in preeclampsia and a potential therapeutic target. Clinical trial registration number: NCT 01736826.Trial registration: ClinicalTrials.gov NCT01736826.

IntroductionCoronavirus disease 2019 (COVID-19) is a respiratory disease triggered by immunopathological mechanisms that cause excessive inflammation and leukocyte dysfunction. Neutrophils play a critical role in the innate immunity and are able to produce neutrophil extracellular traps (NETs: NETosis process) to combat infections. Some NETs markers are increased in patients who died from COVID-19. Recently, the neutrophil fluorescence variable (NEU-SFL), available on certain automated complete blood count (CBC) analyzers, has been correlated with NET formation and may reflect NETosis in patients. Here we evaluate whether NEU-SFL measured after admission of COVID-19 patients is associated with in-hospital survival or death.Patients and methods1,852 patients admitted for severe COVID-19 at Nîmes University Hospital in 2021 were retrospectively included in the study: 1,564 who survived the hospital stay and 288 who did not. The NEU-SFL was obtained on the Sysmex™ XN-10® analyzer and values for survivors and non-survivors were compared. The intra-patient NEU-SFL variations between the hospital entry and the last day of hospitalization were also analyzed (IRB 22.06.01, NCT 05413824).ResultsNon-survivors presented higher NEU-SFL values. NEU-SFL values above the 4th quartile were independently associated with a 2.88-fold risk of death. Furthermore, the difference of NEU-SFL values between the first and the last available data during hospitalization revealed that a decrease in NEU-SFL was associated to survivors and vice versa.ConclusionOur study reinforces the role of neutrophils and NETosis in the pathophysiology and prognosis of COVID-19. Further studies combining NEU-SFL with other NETosis markers could improve the management of COVID-19 patients.

NETosis is an innate immune response occurring after infection or inflammation: activated neutrophils expel decondensed DNA in complex with histones into the extracellular environment in a controlled manner. It activates coagulation and fuels the risk of thrombosis. Human pregnancy is associated with a mild proinflammatory state characterized by circulatory neutrophil activation which is further increased in complicated pregnancies, placenta-mediated complications being associated with an increased thrombotic risk. This aberrant activation leads to an increased release of nucleosomes in the blood flow. The aim of our study was to initially quantify nucleosome-bound histones in normal pregnancy and in placenta-mediated complication counterpart. We analyzed the role of histones on extravillous trophoblast function. Circulating nucleosome-bound histones H3 (Nu.QH3.1, Nu.QH3PanCit, Nu.QH3K27me3) and H4 (Nu.QH4K16Ac) were increased in complicated pregnancies. In vitro using the extravillous cell line HTR-8/SVNeo, we observed that free recombinant H2B, H3, and H4 inhibited migration in wound healing assay, but only H3 also blocked invasion in Matrigel-coated Transwell experiments. H3 and H4 also induced apoptosis, whereas H2B did not. Finally, the negative effects of H3 on invasion and apoptosis could be restored with enoxaparin, a low-molecular-weight heparin (LMWH), but not with aspirin. Different circulating nucleosome-bound histones are increased in complicated pregnancy and this would affect migration, invasion, and induce apoptosis of extravillous trophoblasts. Histones might be part of the link between the risk of thrombosis and pregnancy complications, with an effect of LMWH on both.

International audience; Placenta-mediated pregnancy complications generate short- and long-term adverse medical outcomes for both the mother and the fetus. Nucleosomes and free DNA (fDNA) have been described in patients suffering from a wide range of inflammatory conditions.Objective: The objective of our study was to compare nucleosomes and fDNA circulating levels during pregnancy and particularly in women developing a placenta-mediated complication according to the subtype (preeclampsia or intrauterine growth restriction) (NCT01736826).Patients/methods: A total of 115 women were prospectively included in the study across three groups: 30 healthy non-pregnant women, 50 with normal pregnancy, and 35 with a complicated pregnancy. Blood samples were taken up to every 4 weeks for several women with normal pregnancy and nucleosomes and fDNA were quantified using enzyme-linked immunosorbent assay and quantitative polymerase chain reaction, respectively.Results: We show that nucleosomes and fDNA concentrations significantly increase during normal pregnancy, with concentrations at delivery differing between the two groups. Interestingly, we show that concentrations differ according to the type of placenta-mediated complications, with higher levels in preeclampsia compared to intrauterine growth restriction.Conclusions: These data suggest that nucleosomes and fDNA may be additional actors participating in placenta-mediated pregnancy complications.

Thrombosis is the most common, most feared complication of paroxysmal nocturnal haemoglobinuria (PNH), a striking example of the effect of uncontrolled complement activation and haemolysis on vascular biology and haemostasis. Uncontrolled complement activation and haemolysis may occur at any site and there is a higher incidence of thrombosis at atypical sites. The mechanisms are highly multifactorial and still not fully understood. Eculizumab (a complement C5 inhibitor) has been observed to reduce the number of thrombotic events and, consequently, it is thought that the signalling pathways that depend on the activation of complement C5 may be involved. Complement inhibition, initially using eculizumab together with anticoagulation, is thus the key to treating and preventing thrombosis. New proximal complement inhibitors targeting complement C3 have also shown promising results with further improvements in the clinical prognosis and quality of life of patients. Screening for PNH in patients with unexplained thrombosis must be systematically considered in well characterized cases.

Nonvascular neurological manifestations of antiphospholipid antibodies (aPLAbs) are emerging and, among these, several neuropsychiatric shymptoms. Psychiatric diseases are gradually being considered as organic illnesses of the brain. The role of blood-brain barrier regulation is under scrutiny, and increased permeability is thought to play a precipitating role. Neuropsychiatric manifestations in women with antiphospholipid syndrome (APS) are suspected of being secondary to direct binding and the effect of aPLAbs on neurons and glial cells once the permeability of the blood-brain barrier has been altered. Placental diseases, sometimes mediated by aPLAbs, are risk factors for schizophrenia in the offspring, and babies born from women with aPLAbs can develop learning disabilities and autism spectrum disorders. Women with APS more often develop mood disorders as time goes by, and diffusion tensor imaging has evidenced subtle changes in their white matter. More data are urgently needed and the therapeutic management remains to be properly planned.

International audience; Adiponectin receptors (ADIPORs) are integral membrane proteins that control glucose and lipid metabolism by mediating, at least in part, a cellular ceramidase activity that catalyses the hydrolysis of ceramide to produce sphingosine and a free fatty acid (FFA). The crystal structures of the two receptor subtypes, ADIPOR1 and ADIPOR2, show a similar overall seven-transmembrane-domain architecture with large unoccupied cavities and a zinc binding site within the seven transmembrane domain. However, the molecular mechanisms by which ADIPORs function are not known. Here we describe the crystal structure of ADIPOR2 bound to a FFA molecule and show that ADIPOR2 possesses intrinsic basal ceramidase activity that is enhanced by adiponectin. We also identify a ceramide binding pose and propose a possible mechanism for the hydrolytic activity of ADIPOR2 using computational approaches. In molecular dynamics simulations, the side chains of residues coordinating the zinc rearrange quickly to promote the nucleophilic attack of a zinc-bound hydroxide ion onto the ceramide amide carbonyl. Furthermore, we present a revised ADIPOR1 crystal structure exhibiting a seven-transmembrane-domain architecture that is clearly distinct from that of ADIPOR2. In this structure, no FFA is observed and the ceramide binding pocket and putative zinc catalytic site are exposed to the inner membrane leaflet. ADIPOR1 also possesses intrinsic ceramidase activity, so we suspect that the two distinct structures may represent key steps in the enzymatic activity of ADIPORs. The ceramidase activity is low, however, and further studies will be required to characterize fully the enzymatic parameters and substrate specificity of ADIPORs. These insights into ADIPOR function will enable the structure-based design of potent modulators of these clinically relevant enzymes.

International audience; Differentiation of skeletal myoblasts into multinucleated myotubes is a multi-step process orchestrated by several signaling pathways. The Rho small G protein family plays critical roles both during myogenesis induction and myoblast fusion. We report here that in C2C12 myoblasts, expression of RhoE, an atypical member of this family, increases until the onset of myoblast fusion before resuming its basal level once fusion has occurred. We show that RhoE accumulates in elongated, aligned myoblasts prior to fusion and that its expression is also increased during injury-induced skeletal muscle regeneration. Moreover, although RhoE is not required for myogenesis induction, it is essential for myoblast elongation and alignment before fusion and for M-cadherin expression and accumulation at the cell-cell contact sites. Myoblasts lacking RhoE present with defective p190RhoGAP activation and RhoA inhibition at the onset of myoblast fusion. RhoE interacts also with the RhoA effector Rho-associated kinase (ROCK)I whose activity must be downregulated to allow myoblast fusion. Consistently, we show that pharmacological inactivation of RhoA or ROCK restores myoblast fusion in RhoE-deficient myoblasts. RhoE physiological upregulation before myoblast fusion is responsible for the decrease in RhoA and ROCKI activities, which are required for the fusion process. Therefore, we conclude that RhoE is an essential regulator of myoblast fusion.

Cadherins are transmembrane glycoproteins that mediate Ca2+-dependent homophilic cell–cell adhesion and play crucial role during skeletal myogenesis. M-cadherin is required for myoblast fusion into myotubes, but its mechanisms of action remain unknown. The goal of this study was to cast some light on the nature of the M-cadherin–mediated signals involved in myoblast fusion into myotubes. We found that the Rac1 GTPase activity is increased at the time of myoblast fusion and it is required for this process. Moreover, we showed that M-cadherin–dependent adhesion activates Rac1 and demonstrated the formation of a multiproteic complex containing M-cadherin, the Rho-GEF Trio, and Rac1 at the onset of myoblast fusion. Interestingly, Trio knockdown efficiently blocked both the increase in Rac1-GTP levels, observed after M-cadherin–dependent contact formation, and myoblast fusion. We conclude that M-cadherin–dependent adhesion can activate Rac1 via the Rho-GEF Trio at the time of myoblast fusion.

Skeletal muscle retains a resident stem cell population called satellite cells. Although mitotically quiescent in mature muscle, satellite cells can be activated to produce myoblast progeny to generate myonuclei for skeletal muscle homoeostasis, hypertrophy and repair. Regulation of satellite cell function in adult requires redeployment of many of the regulatory networks fundamental to developmental myogenesis. Involved in such control of muscle stem cell fate in embryos are members of the Pitx gene family of bicoid-class homeodomain proteins. Here, we investigated the expression and function of all three Pitx genes in muscle satellite cells of adult mice. Endogenous Pitx1 was undetectable, whilst Pitx2a, Pitx2b and Pitx2c were at low levels in proliferating satellite cells, but increased during the early stages of myogenic differentiation. By contrast, proliferating satellite cells expressed robust amounts of Pitx3, with levels then decreasing as cells differentiated, although Pitx3 remained expressed in unfused myoblasts. To examine the role of Pitx genes in satellite cell function, retroviral-mediated expression of Pitx1, all Pitx2 isoforms or Pitx3, was used. Constitutive expression of any Pitx isoform suppressed satellite cell proliferation, with the cells undergoing enhanced myogenic differentiation. Conversely, myogenic differentiation into multinucleated myotubes was decreased when Pitx2 or Pitx3 levels were reduced using siRNA. Together, our results show that Pitx genes play a role in regulating satellite cell function during myogenesis in adult.

The Rho family of small GTPases are signalling molecules involved in cytoskeleton remodelling and gene transcription. Their activities are important for many cellular processes, including myogenesis. In particular, RhoA positively regulates skeletal-muscle differentiation. We report in the present study that the active form of RhoA increases the expression of utrophin, the autosomal homologue of dystrophin in the mouse C2C12 and rat L8 myoblastic cell lines. Even though this RhoA-dependent utrophin increase is higher in proliferating myoblasts, it is maintained during myogenic differentiation. This occurs via two mechanisms: (i) transcriptional activation of the utrophin promoter A and (ii) post-translational stabilization of utrophin. In addition, RhoA increases plasma-membrane localization of utrophin. Thus RhoA activation up-regulates utrophin levels and enhances its localization at the plasma membrane.