Angelman syndrome (AS) is a severe neurodevelopmental disorder (NDD) caused by loss of functional ubiquitin protein ligase E3A (UBE3A). Previous studies showed that UBE3A plays an important role in the first postnatal weeks of mouse brain development, but its precise role is unknown. Since impaired striatal maturation has been implicated in several mouse models for NDDs, we studied the importance of UBE3A in striatal maturation. We used inducible Ube3a mouse models to investigate the maturation of medium spiny neurons (MSNs) from dorsomedial striatum. MSNs of mutant mice matured properly till postnatal day 15 (P15) but remained hyperexcitable with fewer excitatory synaptic events at later ages, indicative of stalled striatal maturation in Ube3a mice. Reinstatement of UBE3A expression at P21 fully restored MSN excitability but only partially restored synaptic transmission and the operant conditioning behavioral phenotype. Gene reinstatement at P70 failed to rescue both electrophysiological and behavioral phenotypes. In contrast, deletion of Ube3a after normal brain development did not result in these electrophysiological and behavioral phenotypes. This study emphasizes the role of UBE3A in striatal maturation and the importance of early postnatal reinstatement of UBE3A expression to obtain a full rescue of behavioral phenotypes associated with striatal function in AS.
Diana C. Rotaru, Ilse Wallaard, Maud de Vries, Julia van der Bie, Ype Elgersma
B cells within secondary lymphoid tissues (SLTs) encompass a diversity of activation states and multiple maturation processes that reflect antigen recognition and transition through the germinal center (GC) reaction, in which mature B cells differentiate into memory and antibody-secreting cells (ASCs). Here, utilizing single-cell RNA-seq, we identify a range of distinct activation and maturation states of tonsillar-derived B cells. In particular, we identify a previously uncharacterized CCL4/CCL3 chemokine-expressing B-cell population with an expression pattern consistent with BCR/CD40 activation. Furthermore, we present a computational method leveraging regulatory network inference and pseudotemporal modeling to identify upstream transcription factor modulation along a GC to ASC axis of transcriptional maturation. Our dataset provides valuable insight into diverse B-cell functional profiles and will be a useful resource for further studies into the B-cell immune compartment.
Diego A. Espinoza, Carole Le Coz, Emylette Cruz Cabrera, Neil Romberg, Amit Bar-Or, Rui Li
Coagulopathy contributes to the majority of deaths and disabilities associated with traumatic brain injury (TBI). Whether neutrophil extracellular traps (NETs) contribute to an abnormal coagulation state in the acute process of TBI remains unknown. Our objectives were to demonstrate the definitive role of NETs in coagulopathy in TBI. We detected NET markers in 128 TBI patients and 34 healthy subjects. Neutrophil-platelet (PLT) aggregates were detected in blood samples from TBI patients and healthy subjects using flow cytometry and staining with CD41 and CD66b. Endothelial cells (ECs) were incubated with isolate NETs and detected the expression of vascular endothelial (VE)-cadherin, syncanden-1, thrombomodulin, von Willebrand factor (VWF), phosphatidylserine (PS) and and tissue factor (TF). In addition, we established a TBI mouse model to detect the potential role of NETs in TBI associated coagulopathy. NETs generation was mediated by High Mobility Group Box 1(HMGB1) from activated platelets and contribute to procoagulant activity in TBI. Furthermore, coculture experiments indicated that NETs damaged the endothelial barrier and caused these cells to assume a procoagulant phenotype. Moreover, the administration of DNase I before or after brain trauma markedly reduced coagulopathy and improved the survival and clinical outcome of mice with TBI.
Jiaqi Jin, Fang Wang, Jiawei Tian, Xinyi Zhao, Jiawei Dong, Nan Wang, Zhihui Liu, Hontao Zhao, Wenqiang Li, Ge Mang, Shaoshan Hu
Ovarian cancer (OvCa) preferentially metastasizes in association with mesothelial cell-lined surfaces. We sought to determine if mesothelial cells are required for OvCa metastasis and detect alterations in mesothelial cell gene expression and cytokine secretion upon interaction with OvCa cells. Using omental samples from patients with high-grade serous OvCa and mouse models with Wt1-driven GFP-expressing mesothelial cells, we validated the intratumoral localization of mesothelial cells during human and mouse OvCa omental metastasis. Removing mesothelial cells ex vivo from human and mouse omenta or in vivo using diphtheria toxin mediated ablation in Msln-Cre mice, significantly inhibited OvCa cell adhesion and colonization. Human ascites induced angiopoietin-like 4 (ANGPTL4) and stanniocalcin 1 (STC1) expression and secretion by mesothelial cells. Inhibition of STC1 or ANGPTL4 via RNAi, obstructed OvCa cell-induced mesothelial cell to mesenchymal transition while inhibition of ANGPTL4 alone obstructed OvCa cell-induced mesothelial cell migration and glycolysis. Inhibition of mesothelial cell ANGPTL4 secretion via RNAi prevented mesothelial cell induced monocyte migration, endothelial cell vessel formation and OvCa cell adhesion, migration, and proliferation. In contrast, inhibition of mesothelial cell STC1 secretion via RNAi prevented mesothelial cell induced endothelial cell vessel formation and OvCa cell adhesion, migration, proliferation, and invasion. Additionally, blocking ANPTL4 function with antibodies reduced the ex vivo colonization of three different OvCa cell lines on human omental tissue explants and in vivo colonization of ID8p53-/-Brca2-/- cells on mouse omenta. These findings indicate that mesothelial cells are important to the initial stages of OvCa metastasis, and that the crosstalk between mesothelial cells and the tumor microenvironment, promotes OvCa metastasis through the secretion of ANGPTL4.
Preety Bajwa, Kasjusz Kordylewicz, Agnes Bilecz, Ricardo R. Lastra, Kristen Wroblewski, Yuval Rinkevich, Ernst Lengyel, Hilary A. Kenny
Arrhythmogenic cardiomyopathy (AC) is a familial heart disease partly caused by impaired desmosome turnover. Thus, stabilization of desmosome integrity may provide potential new treatment options. Desmosomes, apart from cellular cohesion, provide the structural framework of a signaling hub. Here, we investigated the role of the epidermal growth factor receptor (EGFR) in cardiomyocyte cohesion. We inhibited EGFR under physiological and pathophysiological conditions using the murine plakoglobin knockout AC model, in which EGFR was upregulated. EGFR inhibition enhanced cardiomyocyte cohesion. Immunoprecipitation showed an interaction of EGFR and desmoglein 2 (DSG2). Immunostaining and AFM revealed enhanced DSG2 localization and binding at cell borders upon EGFR inhibition. Enhanced area composita length and desmosome assembly were observed upon EGFR inhibition, confirmed by enhanced DSG2 and desmoplakin (DP) recruitment to cell borders. PamGene Kinase assay performed in HL-1 cells treated with Erlotinib, an EGFR inhibitor, revealed upregulation of RhoA associated protein kinase (ROCK). Erlotinib-mediated desmosome assembly and cardiomyocyte cohesion were abolished upon ROCK inhibition. Thus, inhibiting EGFR, thereby stabilizing desmosome integrity via ROCK, might provide new treatment options for AC.
Maria Shoykhet, Orsela Dervishi, Philipp Menauer, Matthias Hiermaier, Sina Moztarzadeh, Colin Osterloh, Ralf J. Ludwig, Tatjana Williams, Brenda Gerull, Stefan Kaab, Sebastian Clauss, Dominik Schüttler, Jens Waschke, Sunil Yeruva
Metastatic progression of epithelial cancers can be associated with epithelial-mesenchymal-transition (EMT) including transcriptional inhibition of E-cadherin (CDH1) expression. Recently, EM plasticity (EMP) and E-cadherin-mediated, cluster-based metastasis and treatment resistance have become more appreciated. However, the mechanisms that maintain E-cadherin expression in this context are less understood. Through studies of inflammatory breast cancer (IBC) and a 3D tumor cell “emboli” culture paradigm, we discovered that COX-2 (PTGS2), a target gene of C/EBPd (CEBPD), or its metabolite prostaglandin E2 (PGE2) promotes protein stability of E-cadherin, β-catenin and p120 catenin through inhibition of GSK3β. The COX-2 inhibitor celecoxib downregulated E-cadherin complex proteins and caused cell death. Co-expression of E-cadherin and COX-2 was seen in breast cancer patients with poor outcome and, along with inhibitory GSK3β phosphorylation, in patient-derived xenografts (PDX) including triple negative breast cancer (TNBC). Celecoxib alone decreased E-cadherin protein expression within xenograft tumors, though CDH1 mRNA levels increased, and reduced circulating tumor cell (CTC) clusters, and in combination with paclitaxel attenuated or regressed lung metastases. This study uncovered a mechanism by which metastatic breast cancer cells can maintain E-cadherin-mediated cell-cell adhesions and cell survival, suggesting that some patients with COX-2+/E-cadherin+ breast cancer may benefit from targeting of the PGE2 signaling pathway.
Kuppusamy Balamurugan, Dipak K. Poria, Saadiya W. Sehareen, Savitri Krishnamurthy, Wei Tang, Lois McKennett, Veena Padmanaban, Kelli Czarra, Andrew J. Ewald, Naoto T. Ueno, Stefan Ambs, Shikha Sharan, Esta Sterneck
Patients with progressive fibrosing interstitial lung diseases (PF-ILDs) carry a poor prognosis and have limited therapeutic options. A hallmark feature is fibroblast resistance to apoptosis, leading to their persistence, accumulation, and excessive deposition of extracellular matrix. A complex balance of the B cell lymphoma 2 (BCL-2) protein family controlling the intrinsic pathway of apoptosis and fibroblast reliance on antiapoptotic proteins has been hypothesized to contribute to this resistant phenotype. Examination of lung tissue from patients with PF-ILD (idiopathic pulmonary fibrosis and silicosis) and mice with PF-ILD (repetitive bleomycin and silicosis) showed increased expression of antiapoptotic BCL-2 family members in α–smooth muscle actin–positive fibroblasts, suggesting that fibroblasts from fibrotic lungs may exhibit increased susceptibility to inhibition of antiapoptotic BCL-2 family members BCL-2, BCL-XL, and BCL-W with the BH3 mimetic ABT-263. We used 2 murine models of PF-ILD to test the efficacy of ABT-263 in reversing established persistent pulmonary fibrosis. Treatment with ABT-263 induced fibroblast apoptosis, decreased fibroblast numbers, and reduced lung collagen levels, radiographic disease, and histologically evident fibrosis. Our studies provide insight into how fibroblasts gain resistance to apoptosis and become sensitive to the therapeutic inhibition of antiapoptotic proteins. By targeting profibrotic fibroblasts, ABT-263 offers a promising therapeutic option for PF-ILDs.
Joseph C. Cooley, Nomin Javkhlan, Jasmine A. Wilson, Daniel G. Foster, Benjamin L. Edelman, Luis A. Ortiz, David A. Schwartz, David W.H. Riches, Elizabeth F. Redente
Vascular calcification (VC) is concomitant with atherosclerosis, yet it remains uncertain why rupture-prone high-risk plaques do not typically show extensive calcification. Intraplaque hemorrhage (IPH) deposits erythrocyte-derived cholesterol enlarging the necrotic core and promoting the high-risk plaque development. Pro-atherogenic CD163+ alternative macrophages engulf hemoglobin-haptoglobin (HH) complexes at IPH sites. However, their role in VC has never been examined. Here we show, in human arteries, the distribution of CD163+ macrophages correlates inversely with VC. In vitro experiments using vascular smooth muscle cells (VSMC) cultured with HH-exposed human macrophages supernatant (M(Hb)) reduced calcification, while arteries from ApoE-/-CD163-/- mice showed greater VC. M(Hb) supernatant-exposed VSMC showed activated NFκB, while blocking NFκB attenuated the anti-calcific effect of M(Hb) on VSMCs. CD163+ macrophages altered VC through NFκB-induced transcription of hyaluronan synthase (HAS), an enzyme which catalyzes the formation of the extracellular matrix glycosaminoglycan, hyaluronan, within VSMCs. M(Hb) supernatants enhanced HAS production in VSMC, while knocking-down HAS attenuated its anti-calcific effect. NFκB blockade in ApoE-/- mice reduced hyaluronan and increased VC. In human arteries, hyaluronan/HAS were increased in areas of CD163+ macrophage presence. Our findings highlight an important mechanism by which CD163+ macrophages inhibit VC through NFκB-induced HAS augmentation and thus promote the high-risk plaques development.
Atsushi Sakamoto, Rika Kawakami, Masayuki Mori, Liang Guo, Ka Hyun Paek, Jose Verdezoto Mosquera, Anne Cornelissen, Saikat Kumar B. Ghosh, Kenji Kawai, Takao Konishi, Raquel Fernandez, Daniela T. Fuller, Weili Xu, Aimee E. Vozenilek, Yu Sato, Hiroyuki Jinnouchi, Sho Torii, Adam W. Turner, Hirokuni Akahori, Salome Kuntz, Craig C. Weinkauf, Parker J. Lee, Robert Kutys, Kathryn Harris, Alfred Lawrence Killey, Christina M. Mayhew, Matthew Ellis, Leah M. Weinstein, Neel V. Gadhoke, Roma Dhingra, Jeremy Ullman, Endale Armelle Dikongue Emene, Maria E. Romero, Frank D. Kolodgie, Clint L. Miller, Renu Virmani, Aloke V. Finn
Persistent symptoms and radiographic abnormalities suggestive of failed lung repair are among the most common symptoms in patients with COVID-19 after hospital discharge. In mechanically ventilated patients with ARDS secondary to SARS-CoV-2 pneumonia, low tidal volumes to reduce ventilator-induced lung injury necessarily elevate blood CO2 levels, often leading to hypercapnia. The role of hypercapnia on lung repair after injury is not completely understood. Here, using a mouse model of hypercapnia exposure, cell lineage-tracing, spatial transcriptomics and 3D-cultures, we show that hypercapnia limits β-catenin signaling in AT2 cells, leading to their reduced proliferative capacity. Hypercapnia alters expression of major Wnts in PDGFRα+-fibroblasts from those maintaining AT2 progenitor activity towards those that antagonize β-catenin signaling thereby limiting progenitor function. Constitutive activation of β-catenin signaling in AT2 cells or treatment of organoid cultures with recombinant WNT3A protein bypasses the inhibitory effects of hypercapnia. Inhibition of AT2 proliferation in hypercapnic patients may contribute to impaired lung repair after injury, preventing sealing of the epithelial barrier, increasing lung flooding, ventilator dependency and mortality.
Laura A. Dada, Lynn C. Welch, Natalia D. Magnani, Ziyou Ren, Hyebin Han, Patricia L. Brazee, Diego Celli, Annette S. Flozak, Anthea Weng, Mariana Maciel Herrerias, Vitalii Kryvenko, István Vadász, Constance E. Runyan, Hiam Abdala-Valencia, Masahiko Shigemura, S. Marina Casalino-Matsuda, Alexander V. Misharin, G.R. Scott Budinger, Cara J. Gottardi, Jacob I. Sznajder
Cystic fibrosis (CF) is characterized by chronic bacterial infections leading to progressive bronchiectasis and respiratory failure. Pseudomonas aeruginosa (Pa) is the predominant opportunistic pathogen infecting the CF airways. The guanine nucleotide exchange factor Vav3 plays a critical role in Pa adhesion to the CF airways by inducing luminal fibronectin deposition that favors bacteria trapping. Here we report that Vav3 overexpression in CF is caused by upregulation of the mRNA-stabilizing protein HuR. We found that HuR accumulates in the cytoplasm of CF airway epithelial cells, binds to and stabilizes Vav3 mRNA. Interestingly, disruption of HuR-Vav3 mRNA interaction improved the CF epithelial integrity, inhibited the formation of the fibronectin-made bacterial docking platforms and prevented Pa adhesion to the CF airway epithelium. These findings indicate that targeting HuR represents a promising anti-adhesive approach in CF to prevent initial stages of Pa infection in a context of emergence of multidrug resistant pathogens.
Mehdi Badaoui, Cyril Sobolewski, Alexandre Luscher, Marc Bacchetta, Thilo Köhler, Christian van Delden, Michelangelo Foti, Marc Chanson
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