Pulmonary artery hypertension (PAH), which is diagnosed by an abnormal increase of blood pressure in the pulmonary artery, can be a severe disease, leading to heart failure. In recent years, medical imaging, such as echocardiography, magnetic resonance imaging (MRI), and computed tomography (CT), has been widely used due to its non-invasive property. Right pulmonary artery (RPA) wall distensibility derived from CT angiography was reported to serve as a reliabile marker for the diagnosis of PAH. This study presented a robust method for automatic segmentation of artery based on CT angiography. The algorithm can be divided into two steps: generation of initial contour and refinement of edge. In the first step, a series of original images at different cardiac phases were thresholded to retrieve appropriate intensity window of vessels, followed by the determination of initial contours by a series of morphological image processing on the binary images with two simple manual initializations. Initial contours without touching can be taken as the final results of segmentation, when others need further refinement of edge. In the second step, the center of vessel was automatically located by an ellipse fitting method and then the ray casting algorithm was applied to search for possible edge. Disconnected segments of edge will be linked to complete the vessel segmentation. Furthermore, cross-sectional areas of arteries at different cardiac phases can be measured and used to obtain distensibility. In this study, artery wall distensibility of patients and healthy subjects was evaluated on four vessels, including aorta, main pulmonary artery, right and left pulmonary artery. In addition, segmentation results of five subjects were compared with those obtained by manual selection to evaluate the reliability of the proposed method.

Background-Pulmonary artery hypertension (PAH) is characterized by progressive vascular changes causing increased pulmonary resistance and eventual right heart failure (HF). It has been suggested that altered myocardial substrate utilization may be associated with right HF, however these changes have not yet been well characterized. The aim of this study was to evaluate in vivo right ventricular (RV) function and RV glucose and fatty acid metabolism in an experimental model of PAH using non-invasive positron emission tomography (PET) imaging and to investigate the effect of a novel endothelin receptor antagonist, Macitentan, on the development of PAH and RV energetics. Methods and Results-Severe PAH was induced in a total of 11 male Sprague-Dawley rats using a single injection of Sugen5416 followed by chronic hypoxia. The rats were then randomized to treatment or no treatment with Macitentan (30 mg/kg daily) Five and eight weeks post injection, substrate utilization was serially assessed with 2-[18F]fluoro-2-deoxyglucose (FDG) and 4-[18F]fluoro-6-thia-heptadecanoate (FTHA) PET scans for glucose and fatty acid metabolism respectively, and reported as a standardized uptake value (SUV). This data was correlated with in vivo functional measurements with echocardiography and multi gated acquisition scans. The Sugen-hypoxia (SuHx) model resulted in an increase in RV FDG uptake over 8 weeks (SUV control: 1.56 ± 0.38, week 5 SuHx: 4.06 ± 1.90, week 8 SuHx: 4.00 ± 1.60, p<0.005 between control and week 5 SuHx). RV FTHA data showed a trend towards increased uptake with onset of PAH at week 5 SuHx (SUV control: 1.50 ± 0.40, week 5 SuHx: 3.06 ± 1.10, p>0.05). Macitentan significantly decreased RV FDG uptake (SUV week 8 SuHx: 4.00 ± 1.60, week 8 SuHx +ERA: 2.54 ± 0.90, p<0.05). This was associated with improved RV ejection fraction (PAH week 8 untreated: 53.15 ± 9.9% vs PAH week 8 treated: 73.22 ± 4.8%, p<0.01) and improved pulmonary artery pressures measured by pulmonary artery acceleration time (PAH week 8 untreated: 17.32 ± 2.30 ms vs. PAH week 8 treated: 24.38 ± 3.90 ms, p<0.001). There was a strong correlation between increased pulmonary artery pressures and increased RV FDG uptake (r=0.87, p=0.001) as well as a significant inverse relationship between improved RV ejection fraction and decreased RV FDG uptake (r=-0.72, p=0.01). Conclusion-PAH is associated with metabolic changes in the RV, characterized by increased glucose uptake and a trend towards increased RV fatty acid uptake with onset of PAH. Macitentan attenuated RV FDG uptake and significantly increased RV function as well as hemodynamics compared to untreated group.

No / We demonstrate the synergistic benefits of using multiple technologies to investigate complex multi-scale biological responses. The combination of reductionist and integrative methodologies can reveal novel insights into mechanisms of action by tracking changes of in vivo phenomena to alterations in protein activity (or vice versa). We have applied this approach to electrical and mechanical remodelling in right ventricular failure caused by monocrotaline-induced pulmonary artery hypertension in rats. We show arrhythmogenic T-wave alternans in the ECG of conscious heart failure animals. Optical mapping of isolated hearts revealed discordant action potential duration (APD) alternans. Potential causes of the arrhythmic substrate; structural remodelling and/or steep APD restitution and dispersion were observed, with specific remodelling of the Right Ventricular Outflow Tract. At the myocyte level, [Ca2+]i transient alternans were observed together with decreased activity, gene and protein expression of the sarcoplasmic reticulum Ca2+-ATPase (SERCA). Computer simulations of the electrical and structural remodelling suggest both contribute to a less stable substrate. Echocardiography was used to estimate increased wall stress in failure, in vivo. Stretch of intact and skinned single myocytes revealed no effect on the Frank-Starling mechanism in failing myocytes. In isolated hearts acute stretch-induced arrhythmias occurred in all preparations. Significant shortening of the early APD was seen in control but not failing hearts. These observations may be linked to changes in the gene expression of candidate mechanosensitive ion channels (MSCs) TREK-1 and TRPC1/6. Computer simulations incorporating MSCs and changes in ion channels with failure, based on altered gene expression, largely reproduced experimental observations.

Yes / We investigated the steepened dynamic action potential duration (APD) restitution of rats with pulmonary artery hypertension (PAH) and right ventricular (RV) failure and tested whether the observed APD restitution properties were responsible for negative mechanical restitution in these myocytes. PAH and RV failure were provoked in male Wistar rats by a single injection of monocrotaline (MCT) and compared with saline-injected animals (CON). Action potentials were recorded from isolated RV myocytes at stimulation frequencies between 1 and 9Hz. Action potential waveforms recorded at 1Hz were used as voltage clamp profiles (action potential clamp) at stimulation frequencies between 1 and 7Hz to evoke rate-dependent currents. Voltage clamp profiles mimicking typical CON and MCT APD restitution were applied and cell shortening simultaneously monitored. Compared with CON myocytes, MCT myocytes were hypertrophied; had less polarized diastolic membrane potentials; had action potentials that were triggered by decreased positive current density and shortened by decreased negative current density; APD was longer and APD restitution steeper. APD90 restitution was unchanged by exposure to the late Na+-channel blocker (5μM) ranolazine or the intracellular Ca2+ buffer BAPTA. Under AP clamp, stimulation frequency-dependent inward currents were smaller inMCTmyocytes and were abolished by BAPTA. In MCT myocytes, increasing stimulation frequency decreased contraction amplitude when depolarization duration was shortened, to mimic APD restitution, but not when depolarization duration was maintained. We present new evidence that the membrane potential of PAH myocytes is less stable than normal myocytes, being more easily perturbed by external currents. These observations can explain increased susceptibility to arrhythmias. We also present novel evidence that negative APD restitution is at least in part responsible for the negative mechanical restitution in PAH myocytes. Thus, our study links electrical restitution remodeling to a defining mechanical characteristic of heart failure, the reduced ability to respond to an increase in demand.

Uncontrolled growth of vascular stem cells as a result of endothelial-mesenchymal transition is considered to cause hyper-proliferative vascular remodeling in severe pulmonary arterial hypertension (PAH) patients. Hyperplastic intimal growth is one of the causes of closure of the lumen of pulmonary arterioles. This abnormal vessel remodeling leads to the progressive increase in pressure of the pulmonary arterioles causing severe PAH; and debilitating harm to patients resulting in mortality from right heart failure. Environmental factors, including polychlorinated biphenyls (PCBs), are considered to be involved in hyper-proliferative vascular remodeling because genetic makeup can only explain about 10% of severe PAH cases. PCB involvement in lung toxicity has received attention because (i) they have been reported to accumulate in the lung; (ii) PCBs produce pathological vascular remodeling in the experimental model; high levels of PCBs are found in human lung tissue; and (iii) epidemiological studies show the association between lung toxicity and PCBs; and prevalence of hypertension and elevated concentrations of particularly PCB153. Recent studies identify PCB153 as one of the largest contributors for total PCB body burden in humans. Our previous studies demonstrated PCB153 mediated vascular endothelial dysfunction and activated the inhibitor of differentiation protein 3 (ID3). ID3 is an important determinant of mitogen and reactive oxygen species-induced G1→S phase cell cycle progression. Although phosphorylation of ID3 increases cell growth by antagonizing the transcription of cell cycle inhibitors, still there is a critical gap in understanding the molecular mechanism(s) of pulmonary proliferative vascular remodeling associated with PCB exposure in humans and the role of the transcription regulator ID3. Our overall objective was to investigate ID3 mediated transcriptional reprogramming as a driver of PCB153-induced pathological proliferative vascular remodeling. Stable ectopic expression of ID3 in lung endothelial cells contributed to endothelial-mesenchymal transition (EndMT), cell proliferation, and cell migration. Using an endothelial spheroid assay, an established method to measure aberrant hyper-proliferation of endothelial cells in PAH patients, we show that stable ectopic expression of ID3 increased the number and size of vascular spheres. ID3 overexpressing cells exposed to environmentally relevant concentrations of PCB153 showed a two-fold increase in cell proliferation as determined by MTT, SRB, and BrdU assays. ID3 overexpressing cells showed the loss of VE-cadherin and gain of MMP9 and vimentin, which are markers of EndMT. PCB153 also increased phosphorylation of ID3 in lung endothelial cells. To determine the molecular mechanism by which ID3 contributes to hyper-proliferative endothelial cells, we investigated ID3 transcriptional reprogramming using ChIP-Seq and RNA-Seq technology. We show here for the first time that ID3 is part of a more general mechanism of transcriptional regulation. Our ChIP-Seq data show that ID3 binds to a subset of approximately 1200 target genes. Comprehensive motif analysis of ChIP-Seq data using the MEME Suite software toolkit revealed that ID3 bound to the GAGAGAGAGA motif sequence on genomic DNA. We also show a significant preference of ID3 binding to motifs associated with transcription factors IRF1, BC11A, IRF4, PRDM1, FOXJ3, SMAD4, ZBTB6, GATA1, and STAT2. Using an integrative approach of ChIP-Seq and RNA-Seq data, we identified 19 genes whose promoter region was bound by ID3 and RNA was differentially expressed in ID3 overexpressing cells. In summary, our data demonstrated that PCB153 and/or ID3 induces proliferation of lung endothelial cells via transcriptional reprogramming. Discoveries from these findings will lay the necessary groundbreaking work for testing the efficacy of ID3 antagonists for the prevention and treatment of pathological vascular remodeling as well as provide a new paradigm by which PCBs may contribute to lung vascular toxicity.

La sclérose systémique (ScS) est une maladie auto-immune dont l’un des principaux auto-anticorps, dirigé contre la protéine centromérique B (CENP-B), est fortement associé à l’hypertension artérielle pulmonaire, l’une des causes majeures de décès dû à la ScS. L’hypertension résulte de l’occlusion progressive des vaisseaux suite à une hyperactivation des cellules musculaires lisses (CML) de la paroi vasculaire. Cependant, les facteurs responsables de ce remodelage vasculaire restent inconnus. Plusieurs études récentes ont démontré que certains auto-antigènes possèdent des fonctions biologiques additionnelles lorsqu'ils se retrouvent dans le milieu extracellulaire. En effet, une fois libérés par nécrose ou apoptose, ces auto-antigènes adoptent une activité biologique qui s'apparente à celles des cytokines et peuvent ainsi participer aux processus normaux de réparation de blessure et/ou acquérir une activité pathogène qui contribue au développement de certaines maladies auto-immunes. Nos résultats suggèrent que la CENP-B peut être ajoutée à cette liste de molécules bifonctionnelles. À l'aide des techniques d'immunofluorescence, d'ELISA cellulaire et de cytométrie en flux, nous avons démontré que la CENP-B se liait spécifiquement à la surface des CML vasculaire de l’artère pulmonaire avec une plus grande affinité pour le phénotype contractile que synthétique. Cette liaison provoquait la migration des cellules ainsi que la sécrétion de cytokines pro-inflammatoires telles que l’interleukine 6 et 8. Les mécanismes par lesquels la protéine exerçait ces effets impliquaient la phosphorylation de FAK et Src ainsi que la voie des MAP kinases, avec ERK1/2 et p38. Des études de signalisation intracellulaire effectuées à l’aide de plusieurs inhibiteurs spécifiques ainsi que des études de désensibilisation nous ont permis d’identifier le récepteur de la CENP-B en plus d’identifier les mécanismes complets de sa signalisation membranaire. Nous avons démontré que la CENP-B se liait de manière spécifique aux CML vasculaire via le récepteur de chémokine 3 (CCR3) pour ensuite transactiver le récepteur EGF, selon un mécanisme métalloprotéase-dépendant qui implique le relargage du HB-EGF. Cette transactivation est un processus important dans l’activation de la voie des MAP kinases ainsi que dans la sécrétion d’IL-8 induite par la CENP-B. Finalement, nous avons démontré que les auto-anticorps anti-CENP-B pouvaient abolir cette cascade de signalisation, empêchant ainsi la CENP-B d’exercer son rôle de cytokine. L’identification de la CENP-B comme ligand du CCR3 ouvre donc plusieurs perspectives quant à l’étude du rôle pathogène des auto-anticorps anti-CENP-B dans la ScS. / CENP-B is a highly conserved, centromere associated protein and is a major autoantigen in systemic sclerosis (SSc). Anti-CENP-B autoantibodies are associated with prominent vascular manifestations such as pulmonary arterial hypertension (PAH) in the limited cutaneous subset of SSc. PAH occurs as a consequence of progressive obliteration of small arteries due to vascular smooth muscle cell dysfunction, migration and proliferation. However, the factors driving this obliteration are unknown. Earlier in vitro studies have demonstrated that some autoantigens have an additional role when they are released in the extracellular environment during the course of injurious insults resulting in cell death. Indeed, it was previously suggested that extracellular autoantigens participate in normal wound repair processes by acting like cytokines and/or chemokines and subsequently displaying pathogenic activities that contribute to the development of autoimmune diseases. Our present findings suggest that the nuclear autoantigen CENP-B can be added to this set of bifunctional molecules. The present study clearly indicates that exogenous CENP-B bound specifically to the surface of human pulmonary artery SMCs. Binding of CENP-B to SMC stimulated their migration during in vitro wound healing assays, as well as their secretion of interleukins 6 and 8. The mechanism by which CENP-B mediated these effects involved the focal adhesion kinase, Src, ERK1/2, and p38 MAPK pathways. Moreover, CENP-B released from apoptotic endothelial cells was found to bind to SMC, thus indicating a plausible in vivo source of extracellular CENP-B. Here, we also report several lines of evidence indicating that CENP-B, which has no obvious primary or secondary structural homology to chemokines, induced SMC activation by interacting with CCR3. Moreover, the present study clearly demonstrates the involvement of EGFR in CENP-B signaling leading to IL-8 secretion. Finally, anti-CENP-B autoantibodies were found to abolish this signaling pathway, thus preventing CENP-B from transactivating EGFR and exerting its cytokine-like activities toward vascular SMCs. The present study sheds new light on the possible role of extracellular CENP-B and its potent biological effects on human pulmonary artery SMCs. The identification of CENP-B as a CCR3 ligand opens up new perspectives for the study of the pathogenic role of anti-CENP-B autoantibodies.

L’intervention de Fontan est une chirurgie palliative pour les cardiopathies congénitales univentriculaire. A long terme, la défaillance cardiaque, l’hypertension artérielle pulmonaire(HTAP) et l’entéropathie exsudative peuvent mener à l’échec tardif du Fontan.Une mise au point clinique a été realisée en se focalisant secondairement sur les patients adultes.L’intérêt de la cavo bipulmonaire (CBP), étape intermédiaire au Fontan, dans certains cas de retours veineux pulmonaires anormaux totaux a été décrite. Afin d’affiner de futures études cliniques, nous avons discuté de l’intérêt du Brain NatriureticPeptide en post opératoire de Fontan. Nous avons démontré la fiabilité d’une nouvelle technologiede l’oxyme´trie de pouls chez des enfants cyanosés et la non fiabilité d’un nouveau dispositifd’impédancemétrie pour le monitorage non invasif du de´bit cardiaque en comparaison avecun cathéter artériel pulmonaire.Un modèle animal viable et chronique de la CBP a été développé. Il a montré que le flux pulsatileantérograde empêche la formation de fistules artério-veineuses pulmonaires (FAVP), améliorel’hématose et atténue le développement de l’HTAP soutenant cliniquement le maintien de ce fluxlors de la création d’une CBP. Par ailleurs, la perte de pulsatilité, en diminuant le contrainte de cisaillement, entraîne unediminution de l’eNOS synthase et une réponse altérée vaso-relaxante dépendante de l'endothélium artérielle pulmonaire. La micropulsatilité limite ces effets délétères. Ces résultats impliquent desthérapies potentielles contre l’HTAP du Fontan, en maintenant un débit pulmonaire accessoire et la modulation pharmaceutique vaso-relaxante non endothélium-dépendante / The Fontan surgery is a palliative surgery for univentricular congenital heart disease. On the longterm, heart failure, pulmonary arterial hypertension (PAH) and exudative enteropathy may lead tothe Fontan failure.A clinical adjustment has been realized by focusing secondarily on adult patients. The advantage, insome cases of total anomalous pulmonary venous return, of the bidirectional cavopulmonary (BCP),which is an intermediate stage of Fontan, has been described.In order to prepare future clinical studies, we have discussed about Brain Natriuretic Peptide inpostoperative Fontan surgery. We have demonstrated the reliability of a new technology of pulseoxymetry in cyanotic children and the unreliability of a new device of impedance for non-invasivemonitoring of cardiac outpout in comparison with pulmonary arterial catheter.A viable chronic animal model of bidirectional cavo-pulmonary (BCP) has been developed. Itshowed that the antegrade pulsatile flow prevents the formation of pulmonary arteriovenous fistulas(PAVMs) ,improves hematosis and attenuates the development of PAH clinically supporting themaintenance of this flow when creating a BCP.Moreover, the loss of pulsatility while reducing the shear stress causes a decrease in e-NOSsynthase and a dependent vaso-relaxing altered response of the pulmonary arterial endothelium. Themicropulsatility limits these deleterious effects. These results imply potential therapies against thePAH of Fontan surgery, maintaining an accessory pulmonary blood flow and the pharmaceuticalvaso-relaxing non-endothelium dependent modulation.

La sclérose systémique (ScS) est une maladie auto-immune dont l’un des principaux auto-anticorps, dirigé contre la protéine centromérique B (CENP-B), est fortement associé à l’hypertension artérielle pulmonaire, l’une des causes majeures de décès dû à la ScS. L’hypertension résulte de l’occlusion progressive des vaisseaux suite à une hyperactivation des cellules musculaires lisses (CML) de la paroi vasculaire. Cependant, les facteurs responsables de ce remodelage vasculaire restent inconnus. Plusieurs études récentes ont démontré que certains auto-antigènes possèdent des fonctions biologiques additionnelles lorsqu'ils se retrouvent dans le milieu extracellulaire. En effet, une fois libérés par nécrose ou apoptose, ces auto-antigènes adoptent une activité biologique qui s'apparente à celles des cytokines et peuvent ainsi participer aux processus normaux de réparation de blessure et/ou acquérir une activité pathogène qui contribue au développement de certaines maladies auto-immunes. Nos résultats suggèrent que la CENP-B peut être ajoutée à cette liste de molécules bifonctionnelles. À l'aide des techniques d'immunofluorescence, d'ELISA cellulaire et de cytométrie en flux, nous avons démontré que la CENP-B se liait spécifiquement à la surface des CML vasculaire de l’artère pulmonaire avec une plus grande affinité pour le phénotype contractile que synthétique. Cette liaison provoquait la migration des cellules ainsi que la sécrétion de cytokines pro-inflammatoires telles que l’interleukine 6 et 8. Les mécanismes par lesquels la protéine exerçait ces effets impliquaient la phosphorylation de FAK et Src ainsi que la voie des MAP kinases, avec ERK1/2 et p38. Des études de signalisation intracellulaire effectuées à l’aide de plusieurs inhibiteurs spécifiques ainsi que des études de désensibilisation nous ont permis d’identifier le récepteur de la CENP-B en plus d’identifier les mécanismes complets de sa signalisation membranaire. Nous avons démontré que la CENP-B se liait de manière spécifique aux CML vasculaire via le récepteur de chémokine 3 (CCR3) pour ensuite transactiver le récepteur EGF, selon un mécanisme métalloprotéase-dépendant qui implique le relargage du HB-EGF. Cette transactivation est un processus important dans l’activation de la voie des MAP kinases ainsi que dans la sécrétion d’IL-8 induite par la CENP-B. Finalement, nous avons démontré que les auto-anticorps anti-CENP-B pouvaient abolir cette cascade de signalisation, empêchant ainsi la CENP-B d’exercer son rôle de cytokine. L’identification de la CENP-B comme ligand du CCR3 ouvre donc plusieurs perspectives quant à l’étude du rôle pathogène des auto-anticorps anti-CENP-B dans la ScS. / CENP-B is a highly conserved, centromere associated protein and is a major autoantigen in systemic sclerosis (SSc). Anti-CENP-B autoantibodies are associated with prominent vascular manifestations such as pulmonary arterial hypertension (PAH) in the limited cutaneous subset of SSc. PAH occurs as a consequence of progressive obliteration of small arteries due to vascular smooth muscle cell dysfunction, migration and proliferation. However, the factors driving this obliteration are unknown. Earlier in vitro studies have demonstrated that some autoantigens have an additional role when they are released in the extracellular environment during the course of injurious insults resulting in cell death. Indeed, it was previously suggested that extracellular autoantigens participate in normal wound repair processes by acting like cytokines and/or chemokines and subsequently displaying pathogenic activities that contribute to the development of autoimmune diseases. Our present findings suggest that the nuclear autoantigen CENP-B can be added to this set of bifunctional molecules. The present study clearly indicates that exogenous CENP-B bound specifically to the surface of human pulmonary artery SMCs. Binding of CENP-B to SMC stimulated their migration during in vitro wound healing assays, as well as their secretion of interleukins 6 and 8. The mechanism by which CENP-B mediated these effects involved the focal adhesion kinase, Src, ERK1/2, and p38 MAPK pathways. Moreover, CENP-B released from apoptotic endothelial cells was found to bind to SMC, thus indicating a plausible in vivo source of extracellular CENP-B. Here, we also report several lines of evidence indicating that CENP-B, which has no obvious primary or secondary structural homology to chemokines, induced SMC activation by interacting with CCR3. Moreover, the present study clearly demonstrates the involvement of EGFR in CENP-B signaling leading to IL-8 secretion. Finally, anti-CENP-B autoantibodies were found to abolish this signaling pathway, thus preventing CENP-B from transactivating EGFR and exerting its cytokine-like activities toward vascular SMCs. The present study sheds new light on the possible role of extracellular CENP-B and its potent biological effects on human pulmonary artery SMCs. The identification of CENP-B as a CCR3 ligand opens up new perspectives for the study of the pathogenic role of anti-CENP-B autoantibodies.