The possible influence of third-order shim coils on gradient-magnet interactions: an inter-field and inter-site study.

OBJECTIVE: To assess the possible influence of third-order shim coils on the behavior of the gradient field and in gradient-magnet interactions at 7 T and above. MATERIALS AND METHODS: Gradient impulse response function measurements were performed at 5 sites spanning field strengths from 7 to 11.7 T, all of them sharing the same exact whole-body gradient coil design. Mechanical fixation and boundary conditions of the gradient coil were altered in several ways at one site to study the impact of mechanical coupling with the magnet on the field perturbations. Vibrations, power deposition in the He bath, and field dynamics were characterized at 11.7 T with the third-order shim coils connected and disconnected inside the Faraday cage. RESULTS: For the same whole-body gradient coil design, all measurements differed greatly based on the third-order shim coil configuration (connected or not). Vibrations and gradient transfer function peaks could be affected by a factor of 2 or more, depending on the resonances. Disconnecting the third-order shim coils at 11.7 T also suppressed almost completely power deposition peaks at some frequencies. DISCUSSION: Third-order shim coil configurations can have major impact in gradient-magnet interactions with consequences on potential hardware damage, magnet heating, and image quality going beyond EPI acquisitions.

Psychosomatic states and their correlation with letting-go: two newly evaluated core factors for investigating the therapeutic effect of spa treatment.

Spa therapy (aka crenobalneotherapy) has been the object of criticism due to insufficient evidence of its effectiveness. While further effectiveness studies are needed, others are also required to better evaluate the curative factors involved during spa therapy that may contribute to the effectiveness. The current study used specific scales to investigate two possible curative factors: the psychosomatic state and the letting-go of patients with mental disorders after 3 weeks of spa therapy. The Saujon Psychosomatic Questionnaire (SPQ) and the Quantified Assessment of Fluidity of Consciousness Questionnaire (QACF) evaluate psychosomatic state and letting-go, respectively. The Hospital Anxiety and Depression Scale (HAD) and the Insomnia Severity Index (ISI) evaluate depression and anxiety symptoms and insomnia symptoms, respectively. Sixty-five subjects (57 women (87.69%); 8 men), with a mean age of 56.9 (± 9.7) years, were included. SPQ and QAFC scores improved significantly before and after the 3 weeks of spa therapy. Improvement in HAD and ISI scores was significantly correlated with SPQ and QACF scores. These preliminary results suggest that the induced psychosomatic state and the letting-go induced by spa treatment with bubbling baths, jet showers, pool bathing and massage could help patients to become more available and more able to change their psychophysiological state.

SOX17 Enhancer Variants Disrupt Transcription Factor Binding And Enhancer Inactivity Drives Pulmonary Hypertension.

BACKGROUND: Pulmonary arterial hypertension (PAH) is a rare disease characterized by remodeling of the pulmonary arteries, increased vascular resistance, and right-sided heart failure. Genome-wide association studies of idiopathic/heritable PAH established novel genetic risk variants, including conserved enhancers upstream of transcription factor (TF) SOX17 containing 2 independent signals. SOX17 is an important TF in embryonic development and in the homeostasis of pulmonary artery endothelial cells (hPAEC) in the adult. Rare pathogenic mutations in SOX17 cause heritable PAH. We hypothesized that PAH risk alleles in an enhancer region impair TF-binding upstream of SOX17, which in turn reduces SOX17 expression and contributes to disturbed endothelial cell function and PAH development. METHODS: CRISPR manipulation and siRNA were used to modulate SOX17 expression. Electromobility shift assays were used to confirm in silico-predicted TF differential binding to the SOX17 variants. Functional assays in hPAECs were used to establish the biological consequences of SOX17 loss. In silico analysis with the connectivity map was used to predict compounds that rescue disturbed SOX17 signaling. Mice with deletion of the SOX17-signal 1 enhancer region (SOX17-4593/enhKO) were phenotyped in response to chronic hypoxia and SU5416/hypoxia. RESULTS: CRISPR inhibition of SOX17-signal 2 and deletion of SOX17-signal 1 specifically decreased SOX17 expression. Electromobility shift assays demonstrated differential binding of hPAEC nuclear proteins to the risk and nonrisk alleles from both SOX17 signals. Candidate TFs HOXA5 and ROR-α were identified through in silico analysis and antibody electromobility shift assays. Analysis of the hPAEC transcriptomes revealed alteration of PAH-relevant pathways on SOX17 silencing, including extracellular matrix regulation. SOX17 silencing in hPAECs resulted in increased apoptosis, proliferation, and disturbance of barrier function. With the use of the connectivity map, compounds were identified that reversed the SOX17-dysfunction transcriptomic signatures in hPAECs. SOX17 enhancer knockout in mice reduced lung SOX17 expression, resulting in more severe pulmonary vascular leak and hypoxia or SU5416/hypoxia-induced pulmonary hypertension. CONCLUSIONS: Common PAH risk variants upstream of the SOX17 promoter reduce endothelial SOX17 expression, at least in part, through differential binding of HOXA5 and ROR-α. Reduced SOX17 expression results in disturbed hPAEC function and PAH. Existing drug compounds can reverse the disturbed SOX17 pulmonary endothelial transcriptomic signature.

PGE2 Supplementation of Oocyte Culture Media Improves the Developmental and Cryotolerance Performance of Bovine Blastocysts Derived From a Serum-Free in vitro Production System, Mirroring the Inner Cell Mass Transcriptome.

The culture media used throughout the in vitro production (IVP) of bovine embryos remain complex. The serum added to culture media in order to improve embryo development negatively impacts the cryotolerance of blastocysts. Periconceptional prostaglandin E2 (PGE2) signaling is known to exert prosurvival effects on in vitro-generated blastocysts. The purpose of the present study was to evaluate the effects on developmental and cryotolerance performance of a serum-free (SF) IVP system that included defined oocyte culture media supplemented or not with PGE2, versus serum-containing (SC) IVP. RNA-sequencing analysis was used to examine the gene expression of ICM derived under the different IVP conditions. We assessed the degree of cryotolerance of grade-I blastocysts during a three-day post-thaw culture by measuring survival and hatching rates, counting trophectoderm and inner cell mass (ICM) blastomere numbers. We also determined the proportion of ICM cells expressing octamer-binding transcription factor 4 protein (OCT4/POU5F1). We showed that grade-I blastocyst development rates under SF + PGE2 conditions were similar to those obtained under SC conditions, although the cleavage rate remained significantly lower. SC IVP conditions induced changes to ICM gene expression relative to several metabolic processes, catabolic activities, cell death and apoptosis. These alterations were associated with significantly higher levels of ICM cell death at day 7 post-fertilization, and lower survival and hatching rates after thawing. SF IVP conditions supplemented or not with PGE2 induced changes to ICM gene expression related to DNA replication, metabolism and double-strand break repair processes, and were associated with significantly larger ICM cell populations after thawing. SF + PGE2 IVP induced changes to ICM gene expression related to epigenetic regulation and were associated with a significantly higher proportion of ICM cells expressing OCT4. For the first time, our study thus offers a comprehensive analysis of the ICM transcriptome regulated by IVP culture conditions in terms of the cellular changes revealed during culture for three days after thawing.

Analysis of blood parameters and molecular endometrial markers during early reperfusion in two ovine models of uterus transplantation.

The dissection of the veins is the trickiest step of Uterine transplantation (UTx). Performing the anastomosis of a single uterine vein could bring a therapeutic benefit and simplification of surgery and serve for managing unilateral venous thromboses. The objectives of this project were to evaluate the expression of early markers of ischemia-reperfusion and to compare findings following one or two vein anastomoses. Orthotopic uterine auto-transplantations were performed on an ovine model with anastomosis of either two (group 1) or one utero-ovarian veins (group 2). Blood gases, histology and ischemia- reperfusion markers transcripts (PTGS2, IL6, IL8, SOD2, C3, BAX/BCL2 and TLR4) were analyzed as well as PTGS2 protein expression using Western Blot and fluorescence immunolocalization on endometrial biopsies after 3h of reperfusion. Ten ewes were included in the experimentation, 4 were in group1, 3 in group 2, the others being sham operated controls. No significant differences were observed between the two phenotypes. Based on these results, the anastomosis of one single uterine vein appears to be an approach consistent with short-term graft survival. Further experiments will be needed to confirm the reliability of this approach, especially the long-term follow-up of the uterine graft including its ability to support gestation to term.

Characterization of acute TLR-7 agonist-induced hemorrhagic myocarditis in mice by multiparametric quantitative cardiac magnetic resonance imaging.

Hemorrhagic myocarditis is a potentially fatal complication of excessive levels of systemic inflammation. It has been reported in viral infection, but is also possible in systemic autoimmunity. Epicutaneous treatment of mice with the Toll-like receptor 7 (TLR-7) agonist Resiquimod induces auto-antibodies and systemic tissue damage, including in the heart, and is used as an inducible mouse model of systemic lupus erythematosus (SLE). Here, we show that overactivation of the TLR-7 pathway of viral recognition by Resiquimod treatment of CFN mice induces severe thrombocytopenia and internal bleeding, which manifests most prominently as hemorrhagic myocarditis. We optimized a cardiac magnetic resonance (CMR) tissue mapping approach for the in vivo detection of diffuse infiltration, fibrosis and hemorrhages using a combination of T1, T2 and T2* relaxation times, and compared results with ex vivo histopathology of cardiac sections corresponding to CMR tissue maps. This allowed detailed correlation between in vivo CMR parameters and ex vivo histopathology, and confirmed the need to include T2* measurements to detect tissue iron for accurate interpretation of pathology associated with CMR parameter changes. In summary, we provide detailed histological and in vivo imaging-based characterization of acute hemorrhagic myocarditis as an acute cardiac complication in the mouse model of Resiquimod-induced SLE, and a refined CMR protocol to allow non-invasive longitudinal in vivo studies of heart involvement in acute inflammation. We propose that adding T2* mapping to CMR protocols for myocarditis diagnosis improves diagnostic sensitivity and interpretation of disease mechanisms.This article has an associated First Person interview with the first author of the paper.

3'-Deoxy-3'-[18F]Fluorothymidine Positron Emission Tomography Depicts Heterogeneous Proliferation Pathology in Idiopathic Pulmonary Arterial Hypertension Patient Lung.

BACKGROUND: Pulmonary vascular cell hyperproliferation is characteristic of pulmonary vascular remodeling in pulmonary arterial hypertension. A noninvasive imaging biomarker is needed to track the pathology and assess the response to novel treatments targeted at resolving the structural changes. Here, we evaluated the application of radioligand 3'-deoxy-3'-[18F]-fluorothymidine (18FLT) using positron emission tomography. METHODS AND RESULTS: We performed dynamic 18FLT positron emission tomography in 8 patients with idiopathic pulmonary arterial hypertension (IPAH) and applied in-depth kinetic analysis with a reversible 2-compartment 4k model. Our results show significantly increased lung 18FLT phosphorylation (k3) in patients with IPAH compared with nonpulmonary arterial hypertension controls (0.086±0.034 versus 0.054±0.009 min-1; P<0.05). There was heterogeneity in the lung 18FLT signal both between patients with IPAH and within the lungs of each patient, compatible with histopathologic reports of lungs from patients with IPAH. Consistent with 18FLT positron emission tomographic data, TK1 (thymidine kinase 1) expression was evident in the remodeled vessels in IPAH patient lung. In addition, hyperproliferative pulmonary vascular fibroblasts isolated from patients with IPAH exhibited upregulated expression of TK1 and the thymidine transporter, ENT1 (equilibrative nucleoside transporter 1). In the monocrotaline and SuHx (Sugen hypoxia) rat pulmonary arterial hypertension models, increased lung 18FLT uptake was strongly associated with peripheral pulmonary vascular muscularization and the proliferation marker, Ki-67 score, together with prominent TK1 expression in remodeled vessels. Importantly, lung 18FLT uptake was attenuated by 2 antiproliferative treatments: dichloroacetate and the tyrosine kinase inhibitor, imatinib. CONCLUSIONS: Dynamic 18FLT positron emission tomography imaging can be used to report hyperproliferation in pulmonary hypertension and merits further study to evaluate response to treatment in patients with IPAH.

Enhanced Anti-lymphoma Activity of CAR19-iNKT Cells Underpinned by Dual CD19 and CD1d Targeting.

Chimeric antigen receptor anti-CD19 (CAR19)-T cell immunotherapy-induced clinical remissions in CD19+ B cell lymphomas are often short lived. We tested whether CAR19-engineering of the CD1d-restricted invariant natural killer T (iNKT) cells would result in enhanced anti-lymphoma activity. CAR19-iNKT cells co-operatively activated by CD1d- and CAR19-CD19-dependent interactions are more effective than CAR19-T cells against CD1d-expressing lymphomas in vitro and in vivo. The swifter in vivo anti-lymphoma activity of CAR19-iNKT cells and their enhanced ability to eradicate brain lymphomas underpinned an improved tumor-free and overall survival. CD1D transcriptional de-repression by all-trans retinoic acid results in further enhanced cytotoxicity of CAR19-iNKT cells against CD19+ chronic lymphocytic leukemia cells. Thus, iNKT cells are a highly efficient platform for CAR-based immunotherapy of lymphomas and possibly other CD1d-expressing cancers.

Tipifarnib prevents development of hypoxia-induced pulmonary hypertension.

Aims: RhoB plays a key role in the pathogenesis of hypoxia-induced pulmonary hypertension. Farnesylated RhoB promotes growth responses in cancer cells and we investigated whether inhibition of protein farnesylation will have a protective effect. Methods and results: The analysis of lung tissues from rodent models and pulmonary hypertensive patients showed increased levels of protein farnesylation. Oral farnesyltransferase inhibitor tipifarnib prevented development of hypoxia-induced pulmonary hypertension in mice. Tipifarnib reduced hypoxia-induced vascular cell proliferation, increased endothelium-dependent vasodilatation and reduced vasoconstriction of intrapulmonary arteries without affecting cell viability. Protective effects of tipifarnib were associated with inhibition of Ras and RhoB, actin depolymerization and increased eNOS expression in vitro and in vivo. Farnesylated-only RhoB (F-RhoB) increased proliferative responses in cultured pulmonary vascular cells, mimicking the effects of hypoxia, while both geranylgeranylated-only RhoB (GG-RhoB), and tipifarnib had an inhibitory effect. Label-free proteomics linked F-RhoB with cell survival, activation of cell cycle and mitochondrial biogenesis. Hypoxia increased and tipifarnib reduced the levels of F-RhoB-regulated proteins in the lung, reinforcing the importance of RhoB as a signalling mediator. Unlike simvastatin, tipifarnib did not increase the expression levels of Rho proteins. Conclusions: Our study demonstrates the importance of protein farnesylation in pulmonary vascular remodelling and provides a rationale for selective targeting of this pathway in pulmonary hypertension.

The zinc transporter ZIP12 regulates the pulmonary vascular response to chronic hypoxia.

The typical response of the adult mammalian pulmonary circulation to a low oxygen environment is vasoconstriction and structural remodelling of pulmonary arterioles, leading to chronic elevation of pulmonary artery pressure (pulmonary hypertension) and right ventricular hypertrophy. Some mammals, however, exhibit genetic resistance to hypoxia-induced pulmonary hypertension. We used a congenic breeding program and comparative genomics to exploit this variation in the rat and identified the gene Slc39a12 as a major regulator of hypoxia-induced pulmonary vascular remodelling. Slc39a12 encodes the zinc transporter ZIP12. Here we report that ZIP12 expression is increased in many cell types, including endothelial, smooth muscle and interstitial cells, in the remodelled pulmonary arterioles of rats, cows and humans susceptible to hypoxia-induced pulmonary hypertension. We show that ZIP12 expression in pulmonary vascular smooth muscle cells is hypoxia dependent and that targeted inhibition of ZIP12 inhibits the rise in intracellular labile zinc in hypoxia-exposed pulmonary vascular smooth muscle cells and their proliferation in culture. We demonstrate that genetic disruption of ZIP12 expression attenuates the development of pulmonary hypertension in rats housed in a hypoxic atmosphere. This new and unexpected insight into the fundamental role of a zinc transporter in mammalian pulmonary vascular homeostasis suggests a new drug target for the pharmacological management of pulmonary hypertension.

Iron homeostasis and pulmonary hypertension: iron deficiency leads to pulmonary vascular remodeling in the rat.

RATIONALE: Iron deficiency without anemia is prevalent in patients with idiopathic pulmonary arterial hypertension and associated with reduced exercise capacity and survival. OBJECTIVES: We hypothesized that iron deficiency is involved in the pathogenesis of pulmonary hypertension and iron replacement is a possible therapeutic strategy. METHODS AND RESULTS: Rats were fed an iron-deficient diet (IDD, 7 mg/kg) and investigated for 4 weeks. Iron deficiency was evident from depleted iron stores (decreased liver, serum iron, and ferritin), reduced erythropoiesis, and significantly decreased transferrin saturation and lung iron stores after 2 weeks IDD. IDD rats exhibited profound pulmonary vascular remodeling with prominent muscularization, medial hypertrophy, and perivascular inflammatory cell infiltration, associated with raised pulmonary artery pressure and right ventricular hypertrophy. IDD rat lungs demonstrated increased expression of hypoxia-induced factor-1α and hypoxia-induced factor-2α, nuclear factor of activated T cells and survivin, and signal transducers and activators of transcription-3 activation, which promote vascular cell proliferation and resistance to apoptosis. Biochemical examination showed reduced mitochondrial complex I activity and mitochondrial membrane hyperpolarization in mitochondria from IDD rat pulmonary arteries. Along with upregulation of the glucose transporter, glucose transporter 1, and glycolytic genes, hk1 and pdk1, lung fluorine-18-labeled 2-fluoro-2-deoxyglucose ligand uptake was significantly increased in IDD rats. The hemodynamic and pulmonary vascular remodeling were reversed by iron replacement (ferric carboxymaltose, 75 mg/kg) and attenuated in the presence of iron deficiency by dichloroacetate and imatinib, 2 putative treatments explored for pulmonary arterial hypertension that target aerobic glycolysis and proliferation, respectively. CONCLUSIONS: These data suggest a major role for iron in pulmonary vascular homeostasis and support the clinical evaluation of iron replacement in patients with pulmonary hypertension.

miR-21/DDAH1 pathway regulates pulmonary vascular responses to hypoxia.

The NOS (nitric oxide synthase) inhibitor ADMA (asymmetric dimethylarginine) contributes to the pathogenesis of pulmonary hypertension. Reduced levels of the enzymes metabolizing ADMA, dimethylarginine dimethylaminohydrolases (DDAH1 and DDAH2) and increased levels of miR-21 are linked to disease pathology, but the mechanisms are not understood. In the present study we assessed the potential role of miR-21 in the regulation of hypoxia-induced changes in ADMA metabolism in vitro and in vivo. Hypoxia inhibited DDAH1 and DDAH2 expression and increased ADMA levels in cultured human pulmonary endothelial cells. In contrast, in human pulmonary smooth muscle cells, only DDAH2 was reduced whereas ADMA levels remained unchanged. Endothelium-specific down-regulation of DDAH1 by miR-21 in hypoxia induced endothelial dysfunction and was prevented by overexpression of DDAH1 and miR-21 blockade. DDAH1, but not DDAH2, mRNA levels were reduced, whereas miR-21 levels were elevated in lung tissues from patients with pulmonary arterial hypertension and mice with pulmonary hypertension exposed to 2 weeks of hypoxia. Hypoxic mice treated with miR-21 inhibitors and DDAH1 transgenic mice showed elevated lung DDAH1, increased cGMP levels and attenuated pulmonary hypertension. Regulation of DDAH1 by miR-21 plays a role in the development of hypoxia-induced pulmonary hypertension and may be of broader significance in pulmonary hypertension.

Evidence for the involvement of type I interferon in pulmonary arterial hypertension.

RATIONALE: Evidence is increasing of a link between interferon (IFN) and pulmonary arterial hypertension (PAH). Conditions with chronically elevated endogenous IFNs such as systemic sclerosis are strongly associated with PAH. Furthermore, therapeutic use of type I IFN is associated with PAH. This was recognized at the 2013 World Symposium on Pulmonary Hypertension where the urgent need for research into this was highlighted. OBJECTIVE: To explore the role of type I IFN in PAH. METHODS AND RESULTS: Cells were cultured using standard approaches. Cytokines were measured by ELISA. Gene and protein expression were measured using reverse transcriptase polymerase chain reaction, Western blotting, and immunohistochemistry. The role of type I IFN in PAH in vivo was determined using type I IFN receptor knockout (IFNAR1(-/-)) mice. Human lung cells responded to types I and II but not III IFN correlating with relevant receptor expression. Type I, II, and III IFN levels were elevated in serum of patients with systemic sclerosis associated PAH. Serum interferon γ inducible protein 10 (IP10; CXCL10) and endothelin 1 were raised and strongly correlated together. IP10 correlated positively with pulmonary hemodynamics and serum brain natriuretic peptide and negatively with 6-minute walk test and cardiac index. Endothelial cells grown out of the blood of PAH patients were more sensitive to the effects of type I IFN than cells from healthy donors. PAH lung demonstrated increased IFNAR1 protein levels. IFNAR1(-/-) mice were protected from the effects of hypoxia on the right heart, vascular remodeling, and raised serum endothelin 1 levels. CONCLUSIONS: These data indicate that type I IFN, via an action of IFNAR1, mediates PAH.

Heterogeneity in lung (18)FDG uptake in pulmonary arterial hypertension: potential of dynamic (18)FDG positron emission tomography with kinetic analysis as a bridging biomarker for pulmonary vascular remodeling targeted treatments.

BACKGROUND: Pulmonary arterial hypertension (PAH) is a disease of progressive vascular remodeling, characterized by dysregulated growth of pulmonary vascular cells and inflammation. A prevailing view is that abnormal cellular metabolism, notably aerobic glycolysis that increases glucose demand, underlies the pathogenesis of PAH. Increased lung glucose uptake has been reported in animal models. Few data exist from patients with PAH. METHODS AND RESULTS: Dynamic positron emission tomography imaging with fluorine-18-labeled 2-fluoro-2-deoxyglucose ((18)FDG) ligand with kinetic analysis demonstrated increased mean lung parenchymal uptake in 20 patients with PAH, 18 with idiopathic PAH (IPAH) (FDG score: 3.27±1.22), and 2 patients with connective tissue disease (5.07 and 7.11) compared with controls (2.02±0.71; P<0.05). Further compartment analysis confirmed increased lung glucose metabolism in IPAH. Lung (18)FDG uptake and metabolism varied within the IPAH population and within the lungs of individual patients, consistent with the recognized heterogeneity of vascular pathology in this disease. The monocrotaline rat PAH model also showed increased lung (18)FDG uptake, which was reduced along with improvements in vascular pathology after treatment with dicholoroacetate and 2 tyrosine kinase inhibitors, imatinib and sunitinib. Hyperproliferative pulmonary vascular fibroblasts isolated from IPAH patients exhibited upregulated glycolytic gene expression, along with increased cellular (18)FDG uptake; both were reduced by dicholoroacetate and imatinib. CONCLUSIONS: Some patients with IPAH exhibit increased lung (18)FDG uptake. (18)FDG positron emission tomography imaging is a tool to investigate the molecular pathology of PAH and its response to treatment.

Specific expression patterns and cell distribution of ancient and modern PAG in bovine placenta during pregnancy.

Pregnancy-associated glycoproteins (PAGs) constitute a multigenic family of aspartic proteinases expressed in the trophoblast of the ruminant placenta. In Bos taurus, this family comprises 21 members segregated into ancient and modern phylogenetic groups. Ancient PAGs have been reported to be synthesized throughout the trophoblastic cell layer whereas modern PAGs are produced by binucleate cells of cotyledons. The aim of this study was to investigate modern and ancient PAGs during gestation in cotyledonary and intercotyledonary tissues. To obtain convincing and innovative results despite the high sequence identity shared between PAGs, we designed specific tools such as amplification primers and antibodies. Using real-time RT-PCR, we described the transcript expression of 16 bovine PAGs. Overall, PAGs are characterized by an increase in their expression during gestation. However, we demonstrated a segregation of modern PAGs in cotyledons and of ancient PAGs in the intercotyledonary chorion, except for the ancient PAG2 expressed in cotyledons. By raising specific antibodies against the modern PAG1 and ancient PAG11 and PAG2, we established the expression kinetics of the proteins using western blotting. Immunohistochemistry showed that PAGs were produced by specific cellular populations: PAG1 by binucleate cells in the whole trophoblastic layer, PAG11 was localized in binucleate cells of the intercotyledonary trophoblast and the chorionic plate of the cotyledon, while PAG2 was produced in mononucleate cells of the internal villi of the cotyledon. These results revealed a highly specific regulation of PAG expression and cell localization as a function of their phylogenetic status, suggesting distinct biological functions within placental tissues.

Role of RhoB in the regulation of pulmonary endothelial and smooth muscle cell responses to hypoxia.

RATIONALE: RhoA and Rho kinase contribute to pulmonary vasoconstriction and vascular remodeling in pulmonary hypertension. RhoB, a protein homologous to RhoA and activated by hypoxia, regulates neoplastic growth and vasoconstriction but its role in the regulation of pulmonary vascular function is not known. OBJECTIVE: To determine the role of RhoB in pulmonary endothelial and smooth muscle cell responses to hypoxia and in pulmonary vascular remodeling in chronic hypoxia-induced pulmonary hypertension. METHODS AND RESULTS: Hypoxia increased expression and activity of RhoB in human pulmonary artery endothelial and smooth muscle cells, coincidental with activation of RhoA. Hypoxia or adenoviral overexpression of constitutively activated RhoB increased actomyosin contractility, induced endothelial permeability, and promoted cell growth; dominant negative RhoB or manumycin, a farnesyltransferase inhibitor that targets the vascular function of RhoB, inhibited the effects of hypoxia. Coordinated activation of RhoA and RhoB maximized the hypoxia-induced stress fiber formation caused by RhoB/mammalian homolog of Drosophila diaphanous-induced actin polymerization and RhoA/Rho kinase-induced phosphorylation of myosin light chain on Ser19. Notably, RhoB was specifically required for hypoxia-induced factor-1α stabilization and for hypoxia- and platelet-derived growth factor-induced cell proliferation and migration. RhoB deficiency in mice markedly attenuated development of chronic hypoxia-induced pulmonary hypertension, despite compensatory expression of RhoA in the lung. CONCLUSIONS: RhoB mediates adaptational changes to acute hypoxia in the vasculature, but its continual activation by chronic hypoxia can accentuate vascular remodeling to promote development of pulmonary hypertension. RhoB is a potential target for novel approaches (eg, farnesyltransferase inhibitors) aimed at regulating pulmonary vascular tone and structure.

Differential expression of the enzymatic system controlling synthesis, metabolism, and transport of PGF2 alpha in human fetal membranes.

The present study investigated the expression of genes and proteins associated with PGF2alpha biosynthesis, catabolism, and transport in matched amnion and choriodecidua of human term placenta. The concentration of PGF2alpha within fetal membranes depends on the balance between complex enzymatic systems responsible for, respectively, its synthesis-by prostaglandin-endoperoxide synthase 2 (PTGS2) and members of the aldo-keto reductase (AKR) family, AKR1C3 and AKR1B1-and its catabolic inactivation-through hydroxy-prostaglandin-dehydrogenase (HPGD). We observed that AKR1C3 shows equal basal expression (mRNA and protein) in choriodecidua and amnion but that AKR1B1 exhibits preferential expression in the choriodecidua. Expression of HPGD and solute carrier organic anion transporter family member 2A1 (SLCO2A1) was found primarily in the choriodecidua. We also evaluated whether an inflammatory environment induced by the gram-negative bacterial endotoxin lipopolysaccharide (LPS) affects expression of each candidate enzymes. The amnion responded to LPS with a small but significant decrease of AKR1B1 mRNA expression. In contrast, we found a significant increase in PTGS2 and AKR1C3 mRNA expression in choriodecidua after LPS challenge, but such regulation was confirmed only at protein levels for PTGS2 and not for AKR1C3. Our results suggest that the choriodecidua appears to be the main tissue, which expresses maximally all the components (synthesis, degradation, and transport) controlling PGF2alpha levels.

Secreted surfactant protein A from fetal membranes induces stress fibers in cultured human myometrial cells.

In the present study, we investigated the ability of human fetal membranes (amnion and choriodecidua) to regulate human maternal uterine cell functions through the secretion of surfactant protein (SP)-A and SP-D at the end of pregnancy. We detected the expression of both SP-A (SP-A1 and SP-A2) and SP-D by quantitative reverse transcription polymerase chain reaction. Immunohistochemistry revealed that human fetal membranes expressed both SP-A and SP-D. By Western blot analysis, we demonstrated that SP-A protein expression was predominant in choriodecidua, whereas the amnion predominantly expressed SP-D. Only the secretion of SP-A was evidenced in the culture supernatants of amnion and choriodecidua explants by immunodot blot and confirmed by Western blot. Exogenous human purified SP-A induced stress fiber formation in cultured human myometrial cells via a pathway involving Rho-kinase. Conditioned medium from choriodecidua and amnion explants mimicked the SP-A effect. Treatment of myometrial cells with SP-A-depleted conditioned medium from choriodecidua or amnion explants failed to change the actin dynamic. These data indicate that SP-A released by human fetal membranes is able to exert a paracrine regulation of F-actin filament organization in myometrial cells.

Balneotherapy versus paroxetine in the treatment of generalized anxiety disorder.

INTRODUCTION: Preliminary studies have suggested that balneotherapy (BT) is an effective and well-tolerated treatment for generalized anxiety disorder (GAD) and psychotropic medication withdrawal syndrome. We carried out a study in 4 spa resorts to assess the efficacy of BT in GAD. METHOD: We compared BT to paroxetine in terms of efficacy and safety in a randomized multicentre study lasting 8 weeks. Patients meeting the diagnostic criteria of GAD (DSM-IV) were recruited. Assessments were conducted using the Hamilton Rating Scale for Anxiety (HAM-A) and other scales, by a specifically trained and independent physician. The primary outcome measure was the change in the total HAM-A score between baseline and week 8. RESULTS: A total of 237 outpatients were enrolled in four centres; 117 were assigned randomly to BT and 120 to paroxetine. The mean change in HAM-A scores showed an improvement in both groups with a significant advantage of BT compared to paroxetine (-12.0 vs -8.7; p<0.001). Remission and sustained response rates were also significantly higher in the BT group (respectively 19% vs 7% and 51% vs 28%). CONCLUSION: BT is an interesting way of treating GAD. Due to its safety profile it could also be tested in resistant forms of generalized anxiety and in patients who do not tolerate or are reluctant to use pharmacotherapies.