Increased fibrosis is a characteristic remodeling response to biomechanical and neurohumoral stress and a determinant of cardiac mechanical and electrical dysfunction in disease. Stress-induced activation of cardiac fibroblasts (CF) is a critical step in the fibrotic response, although the precise sequence of events underlying activation of these critical cells in vivo remain unclear. Here, we test the hypothesis that a βIV-spectrin/STAT3 complex is essential for maintenance of a quiescent phenotype (basal non-activated state) in CFs. We report increased fibrosis, decreased cardiac function, and electrical impulse conduction defects in genetic and acquired mouse models of βIV-spectrin deficiency. Loss of betaIV-spectrin function promotes STAT3 nuclear accumulation and transcriptional activity, altered gene expression and CF activation. Furthermore, we demonstrate that a quiescent phenotype may be restored in βIV-spectrin deficient fibroblasts by expressing a βIV-spectrin fragment including the STAT3-binding domain or through pharmacological STAT3 inhibition. We find that in vivo STAT3 inhibition abrogates fibrosis and cardiac dysfunction in the setting of global βIV-spectrin deficiency. Finally, we demonstrate that fibroblast-specific deletion of βIV-spectrin is sufficient to induce fibrosis and decreased cardiac function. We propose that the βIV-spectrin/STAT3 complex is a determinant of fibroblast phenotype and fibrosis, with implications for remodeling response in cardiovascular disease.
Nehal J. Patel, Drew M. Nassal, Amara D. Greer-Short, Sathya D. Unudurthi, Benjamin W. Scandling, Daniel Gratz, Xianyao Xu, Anuradha Kalyanasundaram, Vadim V. Fedorov, Federica Accornero, Peter J. Mohler, Keith J. Gooch, Thomas J. Hund
The roles of macrophages in orchestrating innate immunity through phagocytosis and T lymphocyte activation have been extensively investigated. Much less understood is the unexpected role of macrophages in direct tumor regression. Tumoricidal macrophages can indeed manifest cancer immunoediting activity in the absence of adaptive immunity. We investigated direct macrophage cytotoxicity in malignant pleural mesothelioma, a lethal cancer that develops from mesothelial cells of the pleural cavity after occupational asbestos exposure. In particular, we analyzed the cytotoxic activity of mouse RAW264.7 macrophages upon cell-cell contact with autologous AB1/AB12 mesothelioma cells. We show that macrophages killed mesothelioma cells by oxeiptosis via a mechanism involving enhancer of zeste homolog 2 (EZH2), a histone H3 lysine 27–specific (H3K27-specific) methyltransferase of the polycomb repressive complex 2 (PRC2). A selective inhibitor of EZH2 indeed impaired RAW264.7-directed cytotoxicity and concomitantly stimulated the PD-1 immune checkpoint. In the immunocompetent BALB/c model, RAW264.7 macrophages pretreated with the EZH2 inhibitor failed to control tumor growth of AB1 and AB12 mesothelioma cells. Blockade of PD-1 engagement restored macrophage-dependent antitumor activity. We conclude that macrophages can be directly cytotoxic for mesothelioma cells independent of phagocytosis. Inhibition of the PRC2 EZH2 methyltransferase reduces this activity because of PD-1 overexpression. Combination of PD-1 blockade and EZH2 inhibition restores macrophage cytotoxicity.
Malik Hamaidia, Hélène Gazon, Clotilde Hoyos, Gabriela Brunsting Hoffmann, Renaud Louis, Bernard Duysinx, Luc Willems
The cellular origins of glomerulosclerosis involve activation of parietal epithelial cells (PECs) and progressive podocyte depletion. While mammalian target of rapamycin–mediated (mTOR-mediated) podocyte hypertrophy is recognized as an important signaling pathway in the context of glomerular disease, the role of podocyte hypertrophy as a compensatory mechanism preventing PEC activation and glomerulosclerosis remains poorly understood. In this study, we show that glomerular mTOR and PEC activation–related genes were both upregulated and intercorrelated in biopsies from patients with focal segmental glomerulosclerosis (FSGS) and diabetic nephropathy, suggesting both compensatory and pathological roles. Advanced morphometric analyses in murine and human tissues identified podocyte hypertrophy as a compensatory mechanism aiming to regulate glomerular functional integrity in response to somatic growth, podocyte depletion, and even glomerulosclerosis — all of this in the absence of detectable podocyte regeneration. In mice, pharmacological inhibition of mTOR signaling during acute podocyte loss impaired hypertrophy of remaining podocytes, resulting in unexpected albuminuria, PEC activation, and glomerulosclerosis. Exacerbated and persistent podocyte hypertrophy enabled a vicious cycle of podocyte loss and PEC activation, suggesting a limit to its beneficial effects. In summary, our data highlight a critical protective role of mTOR-mediated podocyte hypertrophy following podocyte loss in order to preserve glomerular integrity, preventing PEC activation and glomerulosclerosis.
Victor G. Puelles, James W. van der Wolde, Nicola Wanner, Markus W. Scheppach, Luise A. Cullen-McEwen, Tillmann Bork, Maja T. Lindenmeyer, Lukas Gernhold, Milagros N. Wong, Fabian Braun, Clemens D. Cohen, Michelle M. Kett, Christoph Kuppe, Rafael Kramann, Turgay Saritas, Claudia R. van Roeyen, Marcus J. Moeller, Leon Tribolet, Richard Rebello, Yu B.Y. Sun, Jinhua Li, Gerard Müller-Newen, Michael D. Hughson, Wendy E. Hoy, Fermin Person, Thorsten Wiech, Sharon D. Ricardo, Peter G. Kerr, Kate M. Denton, Luc Furic, Tobias B. Huber, David J. Nikolic-Paterson, John F. Bertram
Thyroid hormone (TH) signaling is a universal regulator of metabolism, growth, and development. Here, we show that TH-TH receptor (TH-TR) axis alterations are critically involved in diabetic nephropathy–associated (DN-associated) podocyte pathology, and we identify TRα1 as a key regulator of the pathogenesis of DN. In ZSF1 diabetic rats, T3 levels progressively decreased during DN, and this was inversely correlated with metabolic and renal disease worsening. These phenomena were associated with the reexpression of the fetal isoform TRα1 in podocytes and parietal cells of both rats and patients with DN and with the increased glomerular expression of the TH-inactivating enzyme deiodinase 3 (DIO3). In diabetic rats, TRα1-positive cells also reexpressed several fetal mesenchymal and damage-related podocyte markers, while glomerular and podocyte hypertrophy was evident. In vitro, exposing human podocytes to diabetes milieu typical components markedly increased TRα1 and DIO3 expression and induced cytoskeleton rearrangements, adult podocyte marker downregulation and fetal kidney marker upregulation, the maladaptive cell cycle induction/arrest, and TRα1-ERK1/2–mediated hypertrophy. Strikingly, T3 treatment reduced TRα1 and DIO3 expression and completely reversed all these alterations. Our data show that diabetic stress induces the TH-TRα1 axis to adopt a fetal ligand/receptor relationship pattern that triggers the recapitulation of the fetal podocyte phenotype and subsequent pathological alterations.
Valentina Benedetti, Angelo Michele Lavecchia, Monica Locatelli, Valerio Brizi, Daniela Corna, Marta Todeschini, Rubina Novelli, Ariela Benigni, Carlamaria Zoja, Giuseppe Remuzzi, Christodoulos Xinaris
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.
Shilpa Singh, Catherine A. Vaughan, Christopher Rabender, Ross Mikkelsen, Sumitra Deb, Swati Palit Deb
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.
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
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.
Stefanie Geisler, Ryan A. Doan, Galen C. Cheng, Aysel Cetinkaya-Fisgin, Shay X. Huang, Ahmet Höke, Jeffrey Milbrandt, Aaron DiAntonio
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.
John Eppensteiner, Jean Kwun, Uwe Scheuermann, Andrew Barbas, Alexander T. Limkakeng, Maggie Kuchibhatla, Eric A. Elster, Allan D. Kirk, Jaewoo Lee
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.
Inês Cabrita, Roberta Benedetto, Rainer Schreiber, Karl Kunzelmann
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.
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
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