Cell biology
Abstract
Depletion of epithelial cells after lung injury prompts proliferation and epithelial-mesenchymal transition (EMT) of progenitor cells, which repopulates the lost epithelial layer. To investigate cell proliferative function of human oncoprotein MDM2, we generated mouse models targeting human MDM2 expression in either lung Club or alveolar cells after doxycycline treatment. We report that MDM2 expression in lung Club or alveolar cells activates DNA replication specifically in lung progenitor cells only after chemical- or radiation-induced lung injury irrespective of their p53 status. Activation of DNA replication by MDM2 triggered by injury leads to the proliferation of lung progenitor cells and restoration of the lost epithelial layers. Mouse lung with no mdm2 allele loses their ability to replicate DNA, whereas, loss of one mdm2 allele compromises this function, demonstrating the requirement of endogenous MDM2. We show that the p53-independent ability of MDM2 to activate Akt signaling is essential for initiating DNA replication in lung progenitor cells. Furthermore, MDM2 activates the Notch signaling pathway and expression of EMT markers indicative of epithelial regeneration. This is the first report demonstrating direct p53-independent participation of MDM2 in progenitor cell proliferation and epithelial repair after lung injury, distinct from a p53-degrading anti-apoptotic effect preventing injury.
Authors
Shilpa Singh, Catherine A. Vaughan, Christopher Rabender, Ross Mikkelsen, Sumitra Deb, Swati Palit Deb
Abstract
Perturbations in biomechanical stimuli during cardiac development contribute to congenital cardiac defects such as Hypoplastic Left Heart Syndrome (HLHS). This study sought to identify stretch-responsive pathways involved in cardiac development. microRNA (miRNA)-Sequencing identified miR-486 as being increased in cardiomyocytes exposed to cyclic stretch in vitro (63%, p<0.002). The right ventricles of HLHS patients experience increased stretch and have a trend towards higher miR-486 levels 4.9-fold (p=0.08). Sheep RVs dilated from excessive pulmonary blood flow have 60% more miR-486 vs. control RVs (p<0.05). The left ventricles of newborn mice treated with miR-486 mimic are 16.9%-24.6% larger (p<0.01) and display 2.48 fold increase in cardiomyocyte proliferation (p<0.01). miR-486 treatment decreases FoxO1 and Smad signaling, while increasing the protein levels of Stat1. Stat1 associates with Gata4 and Serum Response Factor (Srf), two key cardiac transcription factors whose protein levels increase in response to miR-486. This is the first report of a stretch-responsive miRNA that increases the growth of the ventricle in vivo.
Authors
Stephan Lange, Indroneal Banerjee, Katrina Carrion, Ricardo Serrano, Louisa Habich, Rebecca Kameny, Luisa Lengenfelder, Nancy Dalton, Rudolph Meili, Emma Börgeson, Kirk Peterson, Marco Ricci, Joy Lincoln, Majid Ghassemian, Jeffrey R. Fineman, Juan C. del Álamo, Vishal Nigam
Abstract
Chemotherapy-induced peripheral neuropathy is one of the most prevalent dose-limiting toxicities of anticancer therapy. Development of effective therapies to prevent chemotherapy-induced neuropathies could be enabled by a mechanistic understanding of axonal breakdown following exposure to neuropathy-causing agents. Here, we reveal the molecular mechanisms underlying axon degeneration induced by 2 widely used chemotherapeutic agents with distinct mechanisms of action: vincristine and bortezomib. We showed previously that genetic deletion of SARM1 blocks vincristine-induced neuropathy and demonstrate here that it also prevents axon destruction following administration of bortezomib in vitro and in vivo. Using cultured neurons, we found that vincristine and bortezomib converge on a core axon degeneration program consisting of nicotinamide mononucleotide NMNAT2, SARM1, and loss of NAD+ but engage different upstream mechanisms that closely resemble Wallerian degeneration after vincristine and apoptosis after bortezomib. We could inhibit the final common axon destruction pathway by preserving axonal NAD+ levels or expressing a candidate gene therapeutic that inhibits SARM1 in vitro. We suggest that these approaches may lead to therapies for vincristine- and bortezomib-induced neuropathies and possibly other forms of peripheral neuropathy.
Authors
Stefanie Geisler, Ryan A. Doan, Galen C. Cheng, Aysel Cetinkaya-Fisgin, Shay X. Huang, Ahmet Höke, Jeffrey Milbrandt, Aaron DiAntonio
Abstract
Multiple organ failure (MOF) is the leading cause of late mortality and morbidity in patients who are admitted to intensive care units (ICUs). However, there is an epidemiologic discrepancy in the mechanism of underlying immunologic derangement dependent on etiology between sepsis and trauma patients in MOF. We hypothesized that damage-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs), while both involved in the development of MOF, contribute differently to the systemic innate immune derangement and coagulopathic changes. We found that DAMPs not only produce weaker innate immune activation than counterpart PAMPs, but also induce less TLR signal desensitization, contribute to less innate immune cell death, and propagate more robust systemic coagulopathic effects than PAMPs. This differential contribution to MOF provides further insight into the contributing factors to late mortality in critically ill trauma and sepsis patients. These findings will help to better prognosticate patients at risk of MOF and may provide future therapeutic molecular targets in this disease process.
Authors
John Eppensteiner, Jean Kwun, Uwe Scheuermann, Andrew Barbas, Alexander T. Limkakeng, Maggie Kuchibhatla, Eric A. Elster, Allan D. Kirk, Jaewoo Lee
Abstract
Inflammatory airway diseases, such as asthma, cystic fibrosis (CF), and chronic obstructive pulmonary disease (COPD), are characterized by mucus hypersecretion and airway plugging. In both CF and asthma, enhanced expression of the Ca2+-activated Cl– channel TMEM16A is detected in mucus-producing club/goblet cells and airway smooth muscle. TMEM16A contributes to mucus hypersecretion and bronchoconstriction, which are both inhibited by blockers of TMEM16A, such as niflumic acid. Here we demonstrate that the FDA-approved drug niclosamide, a potent inhibitor of TMEM16A identified by high-throughput screening, is an inhibitor of both TMEM16A and TMEM16F. In asthmatic mice, niclosamide reduced mucus production and secretion, as well as bronchoconstriction, and showed additional antiinflammatory effects. Using transgenic asthmatic mice, we found evidence that TMEM16A and TMEM16F are required for normal mucus production/secretion, which may be due to their effects on intracellular Ca2+ signaling. TMEM16A and TMEM16F support exocytic release of mucus and inflammatory mediators, both of which are blocked by niclosamide. Thus, inhibition of mucus and cytokine release, bronchorelaxation, and reported antibacterial effects make niclosamide a potentially suitable drug for the treatment of inflammatory airway diseases, such as CF, asthma, and COPD.
Authors
Inês Cabrita, Roberta Benedetto, Rainer Schreiber, Karl Kunzelmann
Abstract
Circulating macrophages recruited to the lung contribute to pulmonary vascular remodeling in various forms of pulmonary hypertension (PH). In this study we investigated a macrophage phenotype characterized by intracellular iron accumulation and expression of antioxidant (HO-1), vasoactive (ET-1), and proinflammatory (IL-6) mediators observed in the lung tissue of deceased sickle cell disease (SCD) patients with diagnosed PH. To this end, we evaluated an established rat model of group 5 PH that is simultaneously exposed to free hemoglobin (Hb) and hypobaric hypoxia (HX). Here, we tested the hypothesis that pulmonary vascular remodeling observed in human SCD with concomitant PH could be replicated and mechanistically driven in our rat model by a similar macrophage phenotype with iron accumulation and expression of a similar mixture of antioxidant (HO-1), vasoactive (ET-1), and inflammatory (IL-6) proteins. Our data suggest phenotypic similarities between pulmonary perivascular macrophages in our rat model and human SCD with PH, indicating a potentially novel maladaptive immune response to concomitant bouts of Hb and HX exposure. Moreover, by knocking out circulating macrophages with gadolinium trichloride (GdCl3), the response to combined Hb and hypobaric HX was significantly attenuated in rats, suggesting a critical role for macrophages in the exacerbation of SCD PH.
Authors
Katherine Redinus, Jin Hyen Baek, Ayla Yalamanoglu, Hye Kyung H. Shin, Radu Moldova, Julie W. Harral, Delaney Swindle, David Pak, Scott K. Ferguson, Rachelle Nuss, Kathryn Hassell, Eva Nozik-Grayck, Andre F. Palmer, Mehdi A. Fini, Vijaya Karoor, Kurt R. Stenmark, Paul W. Buehler, David C. Irwin
Abstract
Wilms’ tumor is the most common type of childhood kidney cancer. To improve risk stratification and identify novel therapeutic targets for patients with Wilms’ tumor, we used high-resolution mass spectrometry proteomics to identify urine tumor markers associated with Wilms’ tumor relapse. We determined the urine proteomes at diagnosis of 49 patients with Wilms’ tumor, non–Wilms’ tumor renal tumors, and age-matched controls, leading to the quantitation of 6520 urine proteins. Supervised analysis revealed specific urine markers of renal rhabdoid tumors, kidney clear cell sarcomas, renal cell carcinomas as well as those detected in patients with cured and relapsed Wilms’ tumor. In particular, urine prohibitin was significantly elevated at diagnosis in patients with relapsed as compared with cured Wilms’ tumor. In a validation cohort of 139 patients, a specific urine prohibitin ELISA demonstrated that prohibitin concentrations greater than 998 ng/mL at diagnosis were significantly associated with ultimate Wilms’ tumor relapse. Immunohistochemical analysis revealed that prohibitin was highly expressed in primary Wilms’ tumor specimens and associated with disease stage. Using functional genetic experiments, we found that prohibitin was required for the growth and survival of Wilms’ tumor cells. Overexpression of prohibitin was sufficient to block intrinsic mitochondrial apoptosis and to cause resistance to diverse chemotherapy drugs, at least in part by dysregulating factors that control apoptotic cytochrome c release from mitochondrial cristae. Thus, urine prohibitin may improve therapy stratification, noninvasive monitoring of treatment response, and early disease detection. In addition, therapeutic targeting of chemotherapy resistance induced by prohibitin dysregulation may offer improved therapies for patients with Wilms’ and other relapsed or refractory tumors.
Authors
Michael V. Ortiz, Saima Ahmed, Melissa Burns, Anton G. Henssen, Travis J. Hollmann, Ian MacArthur, Shehana Gunasekera, Lyvia Gaewsky, Gary Bradwin, Jeremy Ryan, Anthony Letai, Ying He, Arlene Naranjo, Yueh-Yun Chi, Michael LaQuaglia, Todd Heaton, Paolo Cifani, Jeffrey S. Dome, Samantha Gadd, Elizabeth Perlman, Elizabeth Mullen, Hanno Steen, Alex Kentsis
Abstract
Tissue remodeling/fibrosis is a major feature of all fibrotic diseases, including idiopathic pulmonary fibrosis (IPF). It is underpinned by accumulating extracellular matrix (ECM) proteins. Fibulin-1c (Fbln1c) is a matricellular ECM protein associated with lung fibrosis in both humans and mice, and stabilizes collagen formation. Here we discovered that Fbln1c was increased in the lung tissues of IPF patients and experimental bleomycin-induced pulmonary fibrosis. Fbln1c-deficient (–/–) mice had reduced pulmonary remodeling/fibrosis and improved lung function after bleomycin challenge. Fbln1c interacted with fibronectin, periostin and tenascin-c in collagen deposits following bleomycin challenge. In a novel mechanism of fibrosis Fbln1c bound to latent transforming growth factor (TGF)-β binding protein-1 (LTBP1) to induce TGF-β activation, and mediated downstream Smad3 phosphorylation/signaling. This process increased myofibroblast numbers and collagen deposition. Fbln1 and LTBP1 co-localized in lung tissues from IPF patients. Thus, Fbln1c may be a novel driver of TGF-β-induced fibrosis involving LTBP1 and may be an upstream therapeutic target.
Authors
Gang Liu, Marion A. Cooley, Andrew G. Jarnicki, Theo Borghuis, Prema M. Nair, Gavin Tjin, Alan C. Hsu, Tatt Jhong Haw, Michael Fricker, Celeste L. Harrison, Bernadette Jones, Nicole G. Hansbro, Peter A. Wark, Jay C. Horvat, W. Scott Argraves, Brian G. Oliver, Darryl A. Knight, Janette K. Burgess, Philip M. Hansbro
Abstract
Mitochondrial dysfunction characterizes many rare and common age-associated diseases. The biochemical consequences, underlying clinical manifestations, and potential therapeutic targets, remain to be better understood. We tested the hypothesis that lipid dyshomeostasis in mitochondrial disorders goes beyond mitochondrial fatty acid β-oxidation, particularly in liver. This was achieved using comprehensive untargeted and targeted lipidomics in a case-control cohort of patients with Leigh syndrome French-Canadian variant (LSFC), a mitochondrial disease caused by mutations in LRPPRC, and in mice harboring liver-specific inactivation of Lrpprc (H-Lrpprc–/–). We discovered a plasma lipid signature discriminating LSFC patients from controls encompassing lower levels of plasmalogens and conjugated bile acids, which suggest perturbations in peroxisomal lipid metabolism. This premise was reinforced in H-Lrpprc–/– mice, which compared with littermates recapitulated a similar, albeit stronger peroxisomal metabolic signature in plasma and liver including elevated levels of very-long-chain acylcarnitines. These mice also presented higher transcript levels for hepatic markers of peroxisome proliferation in addition to lipid remodeling reminiscent of nonalcoholic fatty liver diseases. Our study underscores the value of lipidomics to unveil unexpected mechanisms underlying lipid dyshomeostasis ensuing from mitochondrial dysfunction herein implying peroxisomes and liver, which likely contribute to the pathophysiology of LSFC, but also other rare and common mitochondrial diseases.
Authors
Matthieu Ruiz, Alexanne Cuillerier, Caroline Daneault, Sonia Deschênes, Isabelle Robillard Frayne, Bertrand Bouchard, Anik Forest, Julie Thompson Legault, The LSFC Consortium, Frederic M. Vaz, John D. Rioux, Yan Burelle, Christine Des Rosiers
Podoplanin neutralization improves cardiac remodeling and function after acute myocardial infarction
Abstract
Podoplanin, a small mucine-type transmembrane glycoprotein, has been recently shown to be expressed by lymphangiogenic, fibrogenic and mesenchymal progenitor cells in the acutely and chronically infarcted myocardium. Podoplanin binds to CLEC-2, a C-type lectin-like receptor 2 highly expressed by CD11bhigh cells following inflammatory stimuli. Why podoplanin expression appears only after organ injury is currently unknown. Here, we characterize the role of podoplanin in different stages of myocardial repair after infarction and propose a podoplanin-mediated mechanism in the resolution of post-MI inflammatory response and cardiac repair. Neutralization of podoplanin led to significant improvements in the left ventricular functions and scar composition in animals treated with podoplanin neutralizing antibody. The inhibition of the interaction between podoplanin and CLEC-2 expressing immune cells in the heart enhances the cardiac performance, regeneration and angiogenesis post MI. Our data indicates that modulating the interaction between podoplanin positive cells with the immune cells after myocardial infarction positively affects immune cell recruitment and may represent a novel therapeutic target to augment post-MI cardiac repair, regeneration and function.
Authors
Maria Cimini, Venkata Naga Srikanth Garikipati, Claudio de Lucia, Zhongjian Cheng, Chunlin Wang, May M. Truongcao, Anna Maria Lucchese, Rajika Roy, Cindy Benedict, David A. Goukassian, Walter J. Koch, Raj Kishore
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