OBJECTIVES:Pulmonary arterial hypertension (PAH) is a rare disease that may be associated with CTD. Anti-fibroblast (AFA) and AECA have been identified in idiopathic and SSc-associated PAH. The aim was to identify autoantibodies discriminating for PAH associated with SLE, MCTD and primary SS, and their target antigens. METHODS:Sera were collected in the French multicentre auto-HTAP study from 86 patients with CTD excluding SSc, including 32 with PAH (PAH+) and 54 without (PAH-). AFA and AECA were identified using one- (1D) and two-dimensional (2D) immunoblots and proteomics. ELISA tests using human recombinant proteins were used to confirm PAH-associated IgG reactivities. RESULTS:PAH+ patients had similar IgG AFA and AECA reactivities in 56.2% and 40.6% of the cases in 1D immunoblots, respectively. In 2D immunoblots, serum IgG pools from SLE patients (n = 14), MCTD (n = 10), SS (n = 9) and 14 healthy controls (n = 1) recognized, respectively, 273 ± 79, 205 ± 77, 109 ± 11 and 109 protein spots in fibroblasts and 189 ± 48, 146 ± 30, 88 ± 33 and 190 protein spots in endothelial cell extracts. Serum IgG from PAH+ patients recognized 39 fibroblast and 34 endothelial cell protein spots that were not recognized by IgG from PAH- patients, including Annexin A5 (ANXA5). Anti-ANXA5 IgG reactivity was significantly higher in PAH+ compared with PAH- patients with MCTD (73% vs 0%, P < 0.001) and SLE (33% vs 0%, P = 0.009). CONCLUSION:Anti-ANXA5 IgG autoantibody reactivity might represent a predictive biomarker for PAH associated with MCTD and SLE.

Over 95% of Polycythemia Vera (PV) patients carry the V617F mutation in the tyrosine kinase Janus kinase 2 (JAK2), resulting in uncontrolled erythroid proliferation and a high risk of thrombosis. Using mass spectrometry, we analyzed the RBC membrane proteome and showed elevated levels of multiple Ca2+ binding proteins as well as endoplasmic-reticulum-residing proteins in PV RBC membranes compared with RBC membranes from healthy individuals. In this study, we investigated the impact of JAK2V617F on (1) calcium homeostasis and RBC ion channel activity and (2) protein expression and sorting during terminal erythroid differentiation. Our data from automated patch-clamp show modified calcium homeostasis in PV RBCs and cell lines expressing JAK2V617F, with a functional impact on the activity of the Gárdos channel that could contribute to cellular dehydration. We show that JAK2V617F could play a role in organelle retention during the enucleation step of erythroid differentiation, resulting in modified whole cell proteome in reticulocytes and RBCs in PV patients. Given the central role that calcium plays in the regulation of signaling pathways, our study opens new perspectives to exploring the relationship between JAK2V617F, calcium homeostasis, and cellular abnormalities in myeloproliferative neoplasms, including cellular interactions in the bloodstream in relation to thrombotic events.

Endothelial cells (ECs) are constantly submitted in vivo to hemodynamical forces derived from the blood circulation, including shear stress (SS). ECs are able to detect SS and consequently adapt their phenotype, thus affecting many endothelial functions. If a plethora of shear stress-regulated molecular networks have been described in peripheral ECs, less is known about the molecular responses of microvascular brain ECs which constitute the blood–brain barrier (BBB). In this work, we investigated the response of human cerebral microvascular ECs to laminar physiological shear stress using the well characterized hCMEC/D3 cell line. Interestingly, we showed that hCMEC/D3 cells responded to shear stress by aligning perpendicularly to the flow direction, contrary to peripheral endothelial cells which aligned in the flow direction. Whole proteomic profiles were compared between hCMEC/D3 cells cultured either in static condition or under 5 or 10 dyn.cm −2 SS for 3 days. 3592 proteins were identified and expression levels were significantly affected for 3% of them upon both SS conditions. Pathway analyses were performed which revealed that most proteins overexpressed by SS refer to the antioxidant defense, probably mediated by activation of the NRF2 transcriptional factor. Regarding down-regulated proteins, most of them participate to the pro-inflammatory response, cell motility and proliferation. These findings confirm the induction of EC quiescence by laminar physiological SS and reveal a strong protective effect of SS on hCMEC/D3 cells, suggesting a similar effect on the BBB. Our results also showed that SS did not significantly increase expression levels nor did it affect the localization of junctional proteins and did not afect either the functional activity of several ABC transporters (P-glycoprotein and MRPs). This work provides new insights on the response of microvascular brain ECs to SS and on the importance of SS for optimizing in vitro BBB models.

translates into increased nucleoplasmic CaTs and may explain disproportionate rise in nucleoplasmic [Ca 2þ ] in failing cardiomyocytes at increased stimulation frequencies.Therefore, a previously unidentified benefit of restoring the mitochondrial Ca 2þ uptake may be normalization of nuclear Ca 2þ signaling and alleviation of altered excitation-transcription, which could be an important therapeutic approach to prevent adverse cardiac remodeling.

The paper tackles issue of solid-state 3D structural analysis by means of matrix assisted laser desorption/ionization mass spectrometry with a view of our innovative stochastic dynamic new simplistic formula D"SD = 2.6388.10−17.(–2). It represents a universal law, which is obeyed by temporal behavior of experimental variable intensity of any analyte peak of ion, measured in solution under soft-ionization electrospray ionization, collision induced dissociation and atmospheric pressure chemical ionization conditions, respectively. The equation connects between so-called stochastic dynamic diffusion parameter, D"SD, and variable intensity, I, determined per any span of scan time of a measurement. So far, it has been proven empirically, that D"SD parameters of MS ions reflect exactly analyte concentration in solution — a claim independently verified via chromatography — and provide exact information about 3D molecular and electronic structure of molecular species in liquid phase. Herein, we urge that this simplistic formula is a fundamental law also valid to MALDI-MS phenomena; thus, providing exact quantitative and 3D molecular structural information about analytes in solid-state; or, mass spectrometric phenomena in condensed phases are governed by one and the same stochastic dynamic law, which is applicable both to quantitative and 3D analyses of analytes. The latter statement underlines a crucial advantage of the functional relation and emphasizes on incapability of other known fundamental MS theories and equation to deal with simultaneous exact quantitative and 3D structural analyses in the two condensed phases. A body of new empirical evidence is directed to favor the view of application of the model equation to MALDI-MS processes, examining guanosine (1) in mixture of N(2),N(2)-dimethyl guanosine, quantitatively and 3D structurally, with respect to different experimental conditions without, and involving different sample matrix components. The ultra-high accuracy mass spectrometric outcomes are treated by chemomerics. The same is true for utilizing high accuracy quantum chemical static and molecular dynamics in order to correlate experimental mass spectrometric with theoretical quantum chemical data on 3D molecular and electronic structures of nucleosides, their fragment species and energetics in gas and condensed phases. There are detailed subtle electronic effects on various tautomers, ionization processes, radical-ion formation MS reactions, chemical rearrangement, charge and (doubly) proton transfer effects; if any.

Background Mitochondrial nucleoside diphosphate kinase (NDPK-D, NME4, NM23-H4) is a multifunctional enzyme mainly localized in the intermembrane space, bound to the inner membrane. Results We constructed loss-of-function mutants of NDPK-D, lacking either NDP kinase activity or membrane interaction and expressed mutants or wild-type protein in cancer cells. In a complementary approach, we performed depletion of NDPK-D by RNA interference. Both loss-of-function mutations and NDPK-D depletion promoted epithelial-mesenchymal transition and increased migratory and invasive potential. Immunocompromised mice developed more metastases when injected with cells expressing mutant NDPK-D as compared to wild-type. This metastatic reprogramming is a consequence of mitochondrial alterations, including fragmentation and loss of mitochondria, a metabolic switch from respiration to glycolysis, increased ROS generation, and further metabolic changes in mitochondria, all of which can trigger pro-metastatic protein expression and signaling cascades. In human cancer, NME4 expression is negatively associated with markers of epithelial-mesenchymal transition and tumor aggressiveness and a good prognosis factor for beneficial clinical outcome. Conclusions These data demonstrate NME4 as a novel metastasis suppressor gene, the first localizing to mitochondria, pointing to a role of mitochondria in metastatic dissemination.

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NME4 (or nucleoside diphosphate kinase D, NDPK-D, NM23-H4) is a mitochondrial protein with multiple key functions in bioenergetics and signaling. The hexameric NME4 is mainly localized in the intermembrane space, bound to the inner membrane. As we showed earlier, NME4 provides GTP to the mitochondrial GTPase OPA1, stimulates respiration, binds to cardiolipin and promotes its inter-membrane transfer to the mitochondrial surface. Here we deleted either its NDP kinase activity or its cardiolipin interaction and expressed these mutants or wild-type protein in HeLa cells that are almost devoid of endogenous NME4. Both mutations independently led to fragmentation of the mitochondrial network and loss of mitochondrial mass, consistent with disturbed GTP-fueling of the pro-fusion GTPase OPA1. NME4 loss-of-function mutants also showed increased ROS generation and a metabolic shift with reduced cellular respiration and increased aerobic glycolysis. The mitochondrial alterations triggered a specific change in cellular protein expression, with down-regulation of several mitochondrial proteins and altered cell signaling. At the cellular level, NME4 loss-of-function manifested in a morphotypic switch, characterized by inhibited intercellular adhesion, and increased migratory and invasive potential. These data suggest that NME4 loss of function leads to primary alterations in mitochondria that trigger retrograde signaling, finally leading to downstream events responsible for pro-invasive reprogramming of the cell.

Quelque soit l'espèce considérée, Cupressus sempervirens (Cups) en Europe, Cryptomeria japonica (Cryj) au Japon ou Juniperus ashei aux USA, l'allergie au pollen de Cupressacées augmente. Le pollen de Cups est impliqué dans les syndromes croisés cyprès/pêche ou cyprès/agrumes par les allergènes de la famille des protéines régulées par la gibbérelline (GRP). L'objectif du travail est de comparer 2 groupes de jeunes patients japonais allergiques au pollen de Cryj avec (n = 22) ou sans (n = 21) allergie aux fruits et étudier l'implication de la GRP du pollen de Cryj : Cry j 7 dans les réactivités croisées pollen/fruit. Sélection de groupes de patients japonais à l'hôpital National Sagahamira au Japon. Analyse allergomique avec immunoblot direct et compétitif, production d'allergènes recombinants et spectrométrie de masse. Un groupe 3 de jeunes patients japonais allergiques aux fruits seulement (n = 19) et un groupe 4 de patients européens allergiques au pollen de Cups (n = 29) constituent des contrôles supplémentaires. Cry j 7 est reconnu par 46 % des patients allergiques au pollen de Cryj et aux fruits (groupe 2) et par aucun des patients allergiques au pollen seulement (groupe 1). Cry j 7 recombinant inhibe la réactivité IgE à deux isoformes de Cry j 7 natif. Cry j 7 et Cup s 7 croisent. Un allergène acide, de faible masse, indépendant des sensibilisations croisées pollen/fruit est mis en évidence dans Cryj : la calmoduline. Les réactivités croisées pollen/fruits au Japon chez de jeunes enfants impliquent la pêche, les agrumes et la pomme. Des GRP allergèniques ont été décrites dans les deux premiers mais pas dans la pomme. La modélisation 3D de différentes GRP révèle des structures très semblables. En général, les différentes GRP croisent entre elles mais des différences subtiles d'affinité IgE pourraient exister. Le groupe 3 non sensibilisé au pollen de cèdre du Japon est allergique à des fruits différents : le kiwi et la banane. Le syndrome croisé pollen/fruit mis en évidence au Japon et impliquant une sensibilisation à Cry j 7, la GRP du pollen de Cryj est semblable à celui décrit en Europe avec Cup s 7, la GRP de pollen de Cups. Les fruits associés sont les mêmes.

Granulomatosis with polyangiitis (GPA) is an autoimmune vasculitis associated with anti-neutrophil-cytoplasmic antibodies (ANCA) against proteinase 3 leading to kidney damage. Neutrophils from those patients have increased expression of membrane proteinase 3 during apoptosis. Here we examined whether neutrophils from patients with GPA have dysregulated protein expressions associated with apoptosis. A global proteomic analysis was performed comparing neutrophils from patients with GPA, with healthy individuals under basal conditions and during apoptosis. At disease onset, the cytosolic proteome of neutrophils of patients with GPA before treatment was significantly different from healthy controls, and this dysregulation was more pronounced following ex vivo apoptosis. Proteins involved in cell death/survival were altered in neutrophils of patients with GPA. Several proteins identified were PR3-binding partners involved in the clearance of apoptotic cells, namely calreticulin, annexin-A1 and phospholipid scramblase 1. These proteins form a platform at the membrane of apoptotic neutrophils in patients with GPA but not healthy individuals and this was associated with the clinical presentation of GPA. Thus, our study shows that neutrophils from patients with GPA have an intrinsic dysregulation in proteins involved in apoptotic cell clearance, which could contribute to the unabated inflammation and autoimmunity in GPA. Hence, harnessing these dysregulated pathways could lead to novel biomarkers and targeted therapeutic opportunities to treat kidney disease.

Activation of platelets and neutrophils in septic shock results in the formation of microvascular clots containing an intricate scaffold of fibrin with neutrophil extracellular traps (NETs) DNA. NETs contain multiple components that might impact endogenous fibrinolysis, resulting in failure to lyse clots in the microcirculation and residual systemic microthrombosis. We propose herein that the reservoir of human neutrophil elastase (HNE) on NETs may directly interfere with the fibrinolytic mechanism via a plasminogen proteolytic pathway. To investigate this mechanism, we constructed fibrin‐NETs matrices by seeding and activating neutrophils onto a fibrin surface and monitored plasminogen activation or degradation. We demonstrate that the elastase activity of HNE‐DNA complexes is protected from inhibition by plasma antiproteases and sustains its ability to degrade plasminogen. Using mass spectrometry proteomic analysis, we identified plasminogen fragments composed of kringle (K) domains (K 1+2+3 , k 1+2+3+4 ) and the serine protease (SP) region (K 5 ‐SP). We further demonstrate that patients with septic shock with disseminated intravascular coagulation have circulating HNE‐DNA complexes, HNE‐derived plasminogen fragments, a low plasminogen concentration, and a reduced capacity to generate plasmin onto fibrin. In conclusion, we show that NETs bearing active HNE‐DNA complexes reduce plasminogen into fragments, thus impairing fibrinolysis by decreasing the local plasminogen concentration, plasminogen binding to fibrin, and localized plasmin formation.—Barbosa da Cruz, D., Helms, J., Aquino, L. R., Stiel, L., Cougourdan, L., Broussard, C., Chafey, P., Riès‐Kautt, M., Meziani, F., Toti, F., Gaussem, P., Anglés‐Cano, E. DNA‐bound elastase of neutrophil extracellular traps degrades plasminogen, reduces plasmin formation, and decreases fibrinolysis: proof of concept in septic shock plasma. FASEB J. 33, 14270‐14280 (2019). www.fasebj.org

X-linked hypophosphatemia (XLH) is a skeletal disease caused by inactivating mutations in the PHEX gene. Mutated or absent PHEX protein/enzyme leads to a decreased serum phosphate level, which cause mineralization defects in the skeleton and teeth (osteomalacia/odontomalacia). It is not yet altogether clear whether these manifestations are caused solely by insufficient circulating phosphate availability for mineralization or also by a direct, local intrinsic effect caused by impaired PHEX activity. Here, we evaluated the local role of PHEX in a 3-dimensional model of extracellular matrix (ECM) mineralization. Dense collagen hydrogels were seeded either with human dental pulp cells from patients with characterized PHEX mutations or with sex- and age-matched healthy controls and cultured up to 24 d using osteogenic medium with standard phosphate concentration. Calcium quantification, micro–computed tomography, and histology with von Kossa staining for mineral showed significantly lower mineralization in XLH cell-seeded scaffolds, using nonparametric statistical tests. While apatitic mineralization was observed along collagen fibrils by electron microscopy in both groups, Raman microspectrometry indicated that XLH cells harboring the PHEX mutation produced less mineralized scaffolds having impaired mineral quality with less carbonate substitution and lower crystallinity. In the XLH cultures, immunoblotting revealed more abundant osteopontin (OPN), dentin matrix protein 1 (DMP1), and matrix extracellular phosphoglycoprotein (MEPE) than controls, as well as the presence of fragments of these proteins not found in controls, suggesting a role for PHEX in SIBLING protein degradation. Immunohistochemistry revealed altered OPN and DMP1 associated with an increased alkaline phosphatase staining in the XLH cultures. These results are consistent with impaired PHEX activity having local ECM effects in XLH. Future treatments for XLH should target both systemic and local manifestations.

Systemic sclerosis (SSc) is characterized by autoimmunity, vasculopathy and fibrosis. Fibrosis is due to an activation of fibroblasts by the transforming growth factor-ß (TGF-ß). This study investigates the proteomic response of SSc fibroblasts to TGF-ß.Skin fibroblasts from diffuse SSc patients and healthy controls (HC) are cultured with or without TGF-ß. Two-dimensional differential in-gel electrophoresis and mass spectrometry (MS) combined with Ingenuity Pathway analysis (IPA) and Panther/David software analyze proteins differentially expressed between groups. Real-time cell analyzer (RTCA) assesses fibroblast proliferation and viability.Two-hundred-and-seventy-nine proteins are differentially expressed between groups. Principal component analysis shows significant differences between groups. IPA shows specific process networks such as actin cytoskeleton and integrin signaling. Panther and David software show predominant biological processes such as cellular and metabolic processes. TGF-ß enhances protein synthesis and protein pathways. IPA and RTCA suggest the involvement of epidermal growth factor receptor (EGFR) and phosphatidylinositol 3 kinase (Pi3K).That the proteome of fibroblasts differs between SSc patients and HC is confirmed, and it is demonstrated that fibroblasts exacerbate their proteomic phenotype upon stimulation with TGF-ß. EGFR and Pi3K are highlighted as proteins of interest in SSc fibroblasts.

Introduction La sclerodermie systemique (ScS) est une maladie auto-immune rare caracterisee par une auto-immunite, une vasculopathie et de la fibrose. La fibrose est due a une activation exageree des fibroblastes par le facteur de croissance TGF-β. Nous avons ici etudie la reponse proteique de fibroblastes dermiques de patients atteints de ScS apres stimulation par le TGF-β. Materiels et methodes Des fibroblastes cutanes de 4 patients atteints de ScS et de 3 sujets sains ont ete cultives en presence ou en absence de TGF-β. L’electrophorese en gel bidimensionnel et la spectrometrie de masse (MS) ont ete utilisees pour identifier les proteines differemment exprimees entre les groupes. Les logiciels Ingenuity Pathway Analysis® (IPA) et Panther® ont ete utilises pour analyser les proteines identifiees. Enfin, l’analyseur de cellules en temps reel xCelligence® (RTCA) a ete utilise pour evaluer la proliferation et la viabilite des fibroblastes afin de valider les proteines d’interet identifiees prealablement. Resultats Nous avons identifie 279 taches proteiques differemment exprimees entre les groupes. L’analyse en composantes principales a revele des differences significatives entre les fibroblastes chez les patients atteints de ScS et HC lorsqu’ils ont ete cultives en presence ou en absence de TGF-β. Le logiciel IPA® a revele des processus specifiques proteiques tels que l’implication dans l’organisation du cytosquelette, la formation d’actine, la signalisation des integrines, le remodelage cellulaire et les jonctions intercellulaires. Le logiciel Panther® a revele des processus biologiques differentiels tels que les processus cellulaires, les processus metaboliques et l’organisation des composants cellulaires. Le TGF-β a augmente la synthese des proteines des fibroblastes impliquees dans le cytosquelette, la formation d’actine et la signalisation des integrines. En utilisant IPA et RTCA, nous avons identifie et valide la participation du recepteur du facteur de croissance epidermique (EGFR) et de la phosphatidylinositol 3 kinase (Pi3 K) dans la proliferation et la viabilite des fibroblastes chez les patients atteints de ScS. Conclusion Nous avons confirme que le profil de proteome des fibroblastes differe entre les patients atteints de ScS et les sujets sains et demontre que les fibroblastes augmentent leur proteome lors de la stimulation par le TGF-β. Nous avons finalement mis en evidence EGFR et Pi3 K comme proteines d’interet des fibroblastes de patients atteints de ScS et pourraient etre considerees comme de potentielles cibles pour inhiber la fibrose dans la ScS.

Abstract Objective The pathophysiology of giant cell arteritis (GCA) and the mechanisms underlying vascular remodeling, are poorly understood. We aimed to compare vascular smooth muscle cells (VSMCs) from patients with GCA and controls by a proteomic and gene expression profile approach and to identify the signaling pathways involved in proliferation. Methods VSMCs were cultured from temporal artery biopsies (TABs) from patients with biopsy-proven GCA (TAB + -GCA), biopsy-negative GCA (TAB − -GCA), and diagnosis other than GCA (GCA-control). VSMCs from normal human aorta (HAoSMC) were used as controls. 2D-differential in-gel electrophoresis and Affymetrix chips were used to compare proteomes and gene expression profiles of VSMCs. Proliferation was assessed by BrdU incorporation assay. TAB + -GCA and GCA-control TABs underwent immunohistochemistry staining for endothelin-1 (ET-1) and receptors ET A R and ET B R. Results We identified 16, 30 and 2 protein spots differentially expressed between TAB + -GCA and GCA-control VSMCs, TAB + -GCA and TAB − -GCA VSMCs and TAB − -GCA and GCA-control VSMCs, respectively (fold change ≥ 1.5 and p ≤ 0.05). Among the 153 proteins differentially expressed between TAB + -GCA and HAoSMC VSMCs, many were linked with ET-1. Genes differentially expressed between TAB + -GCA and GCA-control VSMCs were involved in proliferation. ET-1 was identified as a link between genes of interest. Proliferation was reduced for TAB + -GCA VSMCs on treatment with the endothelin antagonist macitentan and its active metabolite. Patients showing transmural expression of ET-1 in temporal artery lesions received a significantly higher glucocorticoid daily dose after 6-month follow-up. Conclusion Inhibiting the proliferation with macitentan, combined with glucocorticoids, might be a promising therapeutic approach for patients with GCA.