Research
Abstract
To develop a systems biology model of fibrosis progression within the human lung we performed RNAseq and microRNA analysis on 95 samples obtained from 10 idiopathic pulmonary fibrosis (IPF) and 6 control lungs. Extent of fibrosis in each sample was assessed by microCT measured alveolar surface density (ASD) and confirmed by histology. Regulatory gene expression networks were identified using linear mixed-effect models and dynamic regulatory events miner (DREM). Differential gene expression analysis identified a core set of genes increased or decreased before fibrosis was histologically evident that continued to change with advanced fibrosis. DREM generated a systems biology model of fibrosis progression (available at http: www.sb.cs.cmu.edu/IPFReg) that identified progressively divergent gene expression tracks with microRNAs and transcription factors that specifically regulate early or advanced fibrosis. We confirmed model predictions by demonstrating that expression of POU2AF1, previously unassociated with lung disease but proposed by the model as regulator, is increased in B-lymphocytes in IPF lungs and that POU2AF1 knockout mice were protected from bleomycin induced lung fibrosis. Our results reveal distinct regulation of gene expression changes in IPF tissue that remained structurally normal compared with moderate or advanced fibrosis and suggest distinct regulatory mechanisms for each stage.
Authors
John E. McDonough, Farida Ahangari, Qin Li, Siddhartha Jain, Stijn E. Verleden, Jose Herazo-Maya, Milica Vukmirovic, Giuseppe DeIuliis, Argyrios Tzouvelekis, Naoya Tanabe, Fanny Chu, Xiting Yan, Johny Verschakelen, Robert J. Homer, Dimitris V. Manatakis, Junke Zhang, Jun Ding, Karen Maes, Laurens De Sadeleer, Robin Vos, Arne Neyrinck, Panayiotis V. Benos, Ziv Bar-Joseph, Dean Tantin, James C. Hogg, Bart M. Vanaudenaerde, Wim A. Wuyts, Naftali Kaminski
Abstract
Glomerular disease is characterized by proteinuria and glomerulosclerosis, two pathologic features caused by podocyte injury and mesangial cell activation, respectively. However, whether these two events are linked remains elusive. Here, we report that sonic hedgehog (Shh) is the mediator that connects podocyte damage to mesangial activation and glomerulosclerosis. Shh was induced in glomerular podocytes in various models of proteinuric chronic kidney diseases (CKD). However, mesangial cells in the glomeruli, but not podocytes, responded to hedgehog ligand. In vitro, Shh was induced in podocytes after injury and selectively promoted mesangial cell activation and proliferation. In a mini-organ culture of isolated glomeruli, Shh promoted mesangial activation but did not affect the integrity of podocytes. Podocyte-specific ablation of Shh in vivo exhibited no effect on proteinuria after adriamycin injection but hampered mesangial activation and glomerulosclerosis. Consistently, pharmacologic blockade of Shh signaling decoupled proteinuria from glomerulosclerosis. In humans, Shh was upregulated in glomerular podocytes in patients with CKD and its circulating level was associated with glomerulosclerosis but not proteinuria. These studies demonstrate that Shh mechanistically links podocyte injury to mesangial activation in the pathogenesis of glomerular diseases. Our findings also illustrate a crucial role for podocyte-mesangial communication in connecting proteinuria to glomerulosclerosis.
Authors
Dong Zhou, Haiyan Fu, Yang Han, Lu Zhang, Shijia Liu, Lin Lin, Donna B. Stolz, Youhua Liu
Abstract
Targeted therapies and immunotherapy have shown promise in patients with non-small cell lung cancer (NSCLC). However, the majority of patients fail or become resistant to treatment, emphasizing the need for novel treatments. In this study, we confirm the prognostic value of AXL levels in NSCLC and demonstrate potent anti-tumor activity of the AXL-targeting antibody-drug conjugate enapotamab vedotin across different NSCLC subtypes in a mouse clinical trial of human NSCLC. Tumor regression or stasis was observed in 17/61 (28%) of the PDX models, and was associated with AXL mRNA expression levels. Significant single agent activity of enapotamab vedotin was validated in vivo in 9 of 10 AXL-expressing NSCLC xenograft models. In a panel of EGFR-mutant NSCLC cell lines rendered resistant to EGFR inhibitors (EGFRi) in vitro, we observed de novo or increased AXL protein expression concomitant with enapotamab vedotin-mediated cytotoxicity. Enapotamab vedotin also showed anti-tumor activity in vivo in 3 EGFR-mutant, EGFRi-resistant PDX models, including an osimertinib-resistant NSCLC PDX model. In summary, enapotamab vedotin has promising therapeutic potential in NSCLC. The safety and preliminary efficacy of enapotamab vedotin are currently being evaluated in the clinic across multiple solid tumor types, including NSCLC.
Authors
Louise A. Koopman, Mikkel G. Terp, Gijs G. Zom, Maarten L. Janmaat, Kirstine Jacobsen, Elke Gresnigt - Van den Heuvel, Marcel Brandhorst, Ulf Forssmann, Frederik M. de Bree, Nora Pencheva, Andreas Lingnau, Maria A. Zipeto, Paul W.H.I. Parren, Esther C.W. Breij, Henrik J. Ditzel
Abstract
High levels of circulating miR-16 in the serum of multiple myeloma (MM) patients are independently associated with longer survival. Although the tumor suppressor function of intracellular miR-16 in cancer cells, including MM plasma cells (PCs), has been highly elucidated, its extracellular role in maintaining a non-supportive cancer microenvironment has not been fully explored. Here, we show that miR-16 can be actively secreted by MM cells through extracellular vesicles (EVs), and its extracellular and intracellular levels are directly correlated. We also show that EVs isolated from MM patients and from the conditioned media of MM-PCs can differentiate circulating monocytes to M2-tumor supportive macrophages (TAMs) and that the presence of higher levels of extracellular miR-16 counteracts this effect. In agreement with these observations, our data show that miR-16 directly targets the IKKα/β complex of the NF-kB canonical pathway, which is known to play a critical role in polarizing macrophages toward an M2 phenotype. By using a miR-15a-16-1 knockout mouse model, we also show that loss of the miR-16 cluster supports polarization to M2-macrophages. Finally, we demonstrate the therapeutic benefit of miR-16 overexpression in potentiating the anti-MM activity by a proteasome inhibitor in the presence of MM resident bone marrow TAM.
Authors
Jihane Khalife, Jayeeta Ghose, Marianna Martella, Domenico Viola, Alberto Rocci, Estelle Troadec, Cesar Terrazas, Abhay R. Satoskar, Emine Gulsen Gunes, Ada Dona, James F. Sanchez, P. Leif Bergsagel, Marta Chesi, Alex Pozhitkov, Steven Rosen, Guido Marcucci, Jonathan J. Keats, Craig C. Hofmeister, Amrita Krishnan, Enrico Caserta, Flavia Pichiorri
Abstract
Anemia of β-thalassemias is caused by ineffective erythropoiesis and reduced red cell survival. Several lines of evidence indicate that iron/heme restriction is a potential therapeutic strategy for the disease. Glycine is a key initial substrate for heme and globin synthesis. We provide evidence that bitopertin, a glycine transport inhibitor administered orally, improves anemia, reduces hemolysis, diminishes ineffective erythropoiesis, and increases red cell survival in a mouse model of β-thalassemia (Hbbth3/+ mice). Bitopertin ameliorates erythroid oxidant damage as indicated by a reduction in membrane-associated free α–globin chain aggregates, in reactive oxygen species cellular content, in membrane-bound hemichromes and in HRI activation and elF2α phosphorylation. The improvement of β-thalassemic ineffective erythropoiesis is associated with diminished mTOR activation, Rab5, Lamp1, and p62 accumulation, indicating an improved autophagy. Bitopertin also upregulates liver hepcidin and diminishes liver iron overload. The hematologic improvements achieved by bitopertin are blunted by the concomitant administration of the iron chelator deferiprone, suggesting that an excessive restriction of iron availability might negate the beneficial effects of bitopertin. These data provide important and clinically relevant insights into glycine restriction and reduced heme synthesis strategies for the treatment of β-thalassemia.
Authors
Alessandro Matte, Enrica Federti, Michael Winter, Annette Koerner, Anja Harmeier, Norman Mazer, Tomas Tomka, Maria Luisa Di Paolo, Luigia De Falco, Immacolata Andolfo, Elisabetta Beneduce, Achille Iolascon, Alejandra Macias-Garcia, Jane-Jane Chen, Anne Janin, Christophe Leboeuf, Francesco Turrini, Carlo Brugnara, Lucia De Franceschi
Abstract
Platelet inositol hexakisphosphate kinase 1 (IP6K1) has been shown to control systemic inflammation. Herein, we examined if platelets and IP6K1 regulate pancreatic tissue injury via formation of NETs in experimental models of acute pancreatitis (AP) in mice. By use of electron microscopy abundant NET formation was observed in the inflamed pancreas. These NETs contained numerous microparticles (MP) expressing CD41 or Mac-1. Platelet depletion reduced deposition of NET-MP complexes in the inflamed pancreas. Circulating platelet-neutrophil aggregates (PNA) were increased and inhibition of P-selectin not only disrupted PNA formation but also reduced NETs formation in the inflamed pancreas. NETs depleted of MPs had lower capacity to provoke amylase secretion and STAT-3 phosphorylation in acinar cells. Taurocholate-induced NETs formation, inflammation and tissue damage in the pancreas were decreased in IP6K1-deficient mice. Thrombin stimulation of mixtures of wild-type platelets and neutrophils resulted in NETs formation but not when IP6K1-deficient platelets were incubated with wild-type neutrophils. Polyphosphate rescue restored thrombin-induced NET formation in mixtures of IP6K1-deficient platelets and wild-type neutrophils. Platelet IP6K1 regulates NET-MP complex formation in the pancreas of mice during induction of AP. Targeting platelet IP6K1 might useful to decrease NET-dependent pancreatic tissue inflammation and tissue injury in patients with AP.
Authors
Raed Madhi, Milladur Rahman, Dler Taha, Johan Linders, Mohammed Merza, Yongzhi Wang, Matthias Mörgelin, Henrik Thorlacius
Abstract
The red blood cell (RBC) storage lesion is a multi-parametric response that occurs during storage at 4°C, but its impact on transfused patients remains unclear. In studies of the RBC storage lesion, the temperature transition from cold storage to normal body temperature that occurs during transfusion has received limited attention. We hypothesized that multiple deleterious events might occur in this period of increasing temperature. We show dramatic alterations in several properties of therapeutic blood units stored at 4°C after warming them to normal body temperature (37°C), as well as febrile temperature (40°C). In particular, the intracellular content and redox state of nicotinamide adenine dinucleotide phosphate [NADP(H)] were directly affected by post-storage incubation at 37°C, as well as by pro-oxidant storage conditions. Modulation of the NADPH-producing pentose phosphate pathway, but not the prevention of hemoglobin autoxidation by conversion of oxyhemoglobin to carboxyhemoglobin, provided protection against storage-induced alterations in RBCs, demonstrating the central role of NADPH in mitigating increased susceptibility of stored RBCs to oxidative stress. We propose that assessing RBCs oxidative status after restoration of body temperature provides a sensitive tool to detect storage-related alterations, and has the potential to improve the quality of stored RBCs for transfusion.
Authors
Aline Roch, Nicholas J. Magon, Jessica Maire, Cacang Suarna, Anita Ayer, Sophie Waldvogel, Beat. A. Imhof, Mark J. Koury, Roland Stocker, Marc Schapira
Abstract
Islet transplantation can restore lost glycemic control in type 1 diabetes subjects, but is restricted in its clinical application by limiting supplies of islets and the need for heavy immune suppression to prevent rejection. TNFAIP3, encoding the ubiquitin editing enzyme A20, regulates the activation of immune cells by raising NF-κB signalling thresholds. Here we show that increasing A20 expression in allogeneic islet grafts resulted in permanent survival for approximately 45% of recipients, and > 80% survival when combined with subtherapeutic rapamycin. Allograft survival was dependent upon regulatory T cells, was antigen-specific and grafts showed reduced expression of inflammatory factors. Transplantation of islets with A20 containing a loss-of-function variant (I325N) resulted in increased RIPK1 ubiquitination and NF-κB signalling, graft hyper-inflammation and acute allograft rejection. Overexpression of A20 in human islets potently reduced expression of inflammatory mediators with no impact on glucose stimulated insulin secretion. Therapeutic administration of A20 raises inflammatory signalling thresholds to favour immune tolerance and promotes islet allogeneic survival. Clinically this would allow for reduced immunosuppression and support the use of alternate islet sources.
Authors
Nathan W. Zammit, Stacey N. Walters, Karen L. Seeberger, Philip J. O’Connell, Gregory S. Korbutt, Shane T. Grey
Abstract
Mesenchymal stromal/stem cell (MSC) therapy has shown promise in experimental models of idiopathic pulmonary fibrosis (IPF). The aim of this study was to test the therapeutic effects of MSC-extracellular vesicles/exosomes (MEx) in a bleomycin-induced pulmonary fibrosis model and investigate putative mechanisms of action. Exosomes were isolated from media conditioned by human bone marrow MSCs. Adult mice (C57BL/6 strain) were challenged with endotracheal instillation of bleomycin and treated with MEx concurrently or for reversal models, at day 7 or 21. Experimental groups were assessed at day 7 and/or at day 14 or 28. Bleomycin-challenged mice presented with severe septal thickening and prominent fibrosis, and this was effectively prevented or reversed by a single dose of MEx. Furthermore, MEx therapy modulated whole lung macrophage phenotype and shifted the proportion of lung ‘proinflammatory’ classical monocytes, non-classical monocytes and alveolar macrophages to favor the monocyte/macrophage profiles of untreated-control mice. A parallel immunomodulatory effect was demonstrated in the bone marrow. Notably, transplantation of MEx-preconditioned bone marrow-derived monocytes alleviated core features of pulmonary fibrosis and lung inflammation. Proteomic analysis further revealed a signature enriched in non-inflammatory monocyte genes following MEx therapy supporting the immuno-regulatory, anti-inflammatory effect of MEx.We conclude that a bolus dose of MEx prevents and reverts core features of bleomycin-induced pulmonary fibrosis, and that the beneficial actions of MEx may be mediated via systemic modulation of monocyte phenotypes.
Authors
Nahal Mansouri, Gareth R. Willis, Angeles Fernandez-Gonzalez, Monica Reis, Sina Nassiri, Alex Mitsialis, Stella Kourembanas
Abstract
The acute respiratory distress syndrome (ARDS) is an inflammatory lung disorder that frequently complicates critical illness, and most commonly occurs in the setting of sepsis. Although a number of clinical and environmental risk factors for ARDS have been described, not all patients with risk factors develop the syndrome, raising the possibility of genetic underpinnings for ARDS susceptibility. We have previously reported that circulating cell-free hemoglobin (CFH) is elevated during sepsis, and higher levels are associated with worse outcomes. CFH is rapidly scavenged by the plasma protein haptoglobin (Hp). A common HP genetic variant HP2 is unique to humans and represents 60% of the HP allele frequency in populations of European ancestry. The HP2 gene product has reduced ability to inhibit CFH-mediated inflammation and oxidative stress compared to the alternative HP1. We hypothesized that the HP2 variant increases ARDS susceptibility during sepsis when plasma CFH levels are elevated. In a murine model of sepsis with elevated CFH levels, transgenic mice homozygous for Hp2 had increased lung inflammation, pulmonary vascular permeability, lung apoptosis, and mortality compared to mice homozygous for the alternative allele Hp1. We then tested the clinical relevance of our findings in a prospective observational cohort study of 496 septic critically ill adults, and found that the HP2 variant was significantly associated with increased ARDS susceptibility (odds ratio 1.41 per HP2 allele, 95% confidence interval 1.06 – 1.88, P = 0.018) after controlling for clinical risk factors and plasma CFH. This relationship between the HP2 genetic variant and ARDS risk was only seen in patients with elevated plasma CFH levels. These observations identify the HP2 variant as a novel genetic ARDS risk factor during sepsis, and may have important implications in the study and treatment of ARDS.
Authors
V. Eric Kerchberger, Julie A. Bastarache, Ciara M. Shaver, Hiromasa Nagata, J. Brennan McNeil, Stuart R. Landstreet, Nathan D. Putz, Wen-Kuang Yu, Jordan Jesse, Nancy E. Wickersham, Tatiana N. Sidorova, David R. Janz, Chirag R. Parikh, Edward D. Siew, Lorraine B. Ware
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