Loss-of-function mutations in genes encoding contractile proteins have been observed in thoracic aortic aneurysms (TAA). To gain insight into the contribution of contractile protein deficiency in the pathogenesis of TAA, we examined human aneurysm samples. We found multiple contractile gene products deficient in TAA samples, and in particular, expression of SM22α was inversely correlated with aneurysm size. SM22α-deficient mice demonstrated pregnancy-induced aortic dissection, and SM22α deficiency worsened aortic aneurysm in Fbn1C1039G/+ (Marfan) mice, validating this gene product as a TAA effector. We found that repression of SM22α was enforced by increased activity of the methyltransferase EZH2. TGF-β effectors such as SMAD3 were excluded from binding SM22α-encoding chromatin (TAGLN) in TAA samples, while treatment with the EZH2 inhibitor GSK343 improved cytoskeletal architecture and restored SM22α expression. Finally, inhibition of EZH2 improved aortic performance in Fbn1C1039G/+ mice, in association with restoration of contractile protein expression (including SM22α). Together, these data inform our understanding of contractile protein deficiency in TAA and support the pursuit of chromatin modifying factors as therapeutic targets in aortic disease.
Christian L. Lino Cardenas, Chase W. Kessinger, Carolyn MacDonald, Arminder S. Jassar, Eric M. Isselbacher, Farouc A. Jaffer, Mark E. Lindsay
In multiple sclerosis (MS), a demyelinating inflammatory disease of the CNS, and its animal model (experimental autoimmune encephalomyelitis; EAE), circulating immune cells gain access to the CNS across the blood-brain barrier to cause inflammation, myelin destruction, and neuronal damage. Here, we discovered that calnexin, an ER chaperone, is highly abundant in human brain endothelial cells of MS patients. Conversely, mice lacking calnexin exhibited resistance to EAE induction, no evidence of immune cell infiltration into the CNS, and no induction of inflammation markers within the CNS. Furthermore, calnexin deficiency in mice did not alter the development or function of the immune system. Instead, the loss of calnexin led to a defect in brain endothelial cell function that resulted in reduced T cell trafficking across the blood-brain barrier. These findings identify calnexin in brain endothelial cells as a potentially novel target for developing strategies aimed at managing or preventing the pathogenic cascade that drives neuroinflammation and destruction of the myelin sheath in MS.
Joanna Jung, Paul Eggleton, Alison Robinson, Jessica Wang, Nick Gutowski, Janet Holley, Jia Newcombe, Elzbieta Dudek, Amber M. Paul, Douglas Zochodne, Allison Kraus, Christopher Power, Luis B. Agellon, Marek Michalak
Premature infants are at high risk for developing bronchopulmonary dysplasia (BPD), characterized by chronic inflammation and inhibition of lung development, which we have recently identified as being modulated by microRNAs (miRNAs) and alterations in the airway microbiome. Exosomes and exosomal miRNAs may regulate cell differentiation and tissue and organ development. We discovered that tracheal aspirates from infants with severe BPD had increased numbers of, but smaller, exosomes compared with term controls. Similarly, bronchoalveolar lavage fluid from hyperoxia-exposed mice (an animal model of BPD) and supernatants from hyperoxia-exposed human bronchial epithelial cells (in vitro model of BPD) had increased exosomes compared with air controls. Next, in a prospective cohort study of tracheal aspirates obtained at birth from extremely preterm infants, utilizing independent discovery and validation cohorts, we identified unbiased exosomal miRNA signatures predictive of severe BPD. The strongest signal of reduced miR-876-3p in BPD-susceptible compared with BPD-resistant infants was confirmed in the animal model and in vitro models of BPD. In addition, based on our recent discovery of increased Proteobacteria in the airway microbiome being associated with BPD, we developed potentially novel in vivo and in vitro models for BPD combining Proteobacterial LPS and hyperoxia exposure. Addition of LPS led to a larger reduction in exosomal miR 876-3p in both hyperoxia and normoxia compared with hyperoxia alone, thus indicating a potential mechanism by which alterations in microbiota can suppress miR 876-3p. Gain of function of miR 876-3p improved the alveolar architecture in the in vivo BPD model, demonstrating a causal link between miR 876-3p and BPD. In summary, we provide evidence for the strong predictive biomarker potential of miR 876-3p in severe BPD. We also provide insights on the pathogenesis of neonatal lung disease, as modulated by hyperoxia and microbial product–induced changes in exosomal miRNA 876-3p, which could be targeted for future therapeutic development.
Charitharth Vivek Lal, Nelida Olave, Colm Travers, Gabriel Rezonzew, Kalsang Dolma, Alexandra Simpson, Brian Halloran, Zubair Aghai, Pragnya Das, Nirmal Sharma, Xin Xu, Kristopher Genschmer, Derek Russell, Tomasz Szul, Nengjun Yi, J. Edwin Blalock, Amit Gaggar, Vineet Bhandari, Namasivayam Ambalavanan
Traditional pulmonary therapies for cystic fibrosis (CF) target the downstream effects of CF transmembrane conductance regulator (CFTR) dysfunction (the cause of CF). Use of one such therapy, β-adrenergic bronchodilators (such as albuterol), is nearly universal for airway clearance. Conversely, novel modulator therapies restore function to select mutant CFTR proteins, offering a disease-modifying treatment. Recent trials of modulators targeting F508del-CFTR, the most common CFTR mutation, suggest that chronic β-agonist use may undermine clinical modulator benefits. We therefore sought to understand the impact of chronic or excess β-agonist exposure on CFTR activation in human airway epithelium. The present studies demonstrate a greater than 60% reduction in both wild-type and modulator-corrected F508del-CFTR activation following chronic exposure to short- and long-acting β-agonists. This reduction was due to reduced cellular generation of cAMP downstream of the β-2 adrenergic receptor–G protein complex. Our results point towards a posttranscriptional reduction in adenylyl cyclase function as the mechanism of impaired CFTR activation produced by prolonged β-agonist exposure. β-Agonist–induced CFTR dysfunction was sufficient to abrogate VX809/VX770 modulation of F508del-CFTR in vitro. Understanding the clinical relevance of our observations is critical for CF patients using these drugs, and for investigators to inform future CFTR modulator drug trials.
John J. Brewington, Jessica Backstrom, Amanda Feldman, Elizabeth L. Kramer, Jessica D. Moncivaiz, Alicia J. Ostmann, Xiaoting Zhu, L. Jason Lu, John P. Clancy
Renal fibrosis is a common pathogenic response to injury in chronic kidney disease (CKD). The receptor-interacting protein kinase-3 (RIPK3), a regulator of necroptosis, has been implicated in disease pathogenesis. In mice subjected to unilateral ureteral obstruction–induced (UUO-induced) or adenine diet–induced (AD-induced) renal fibrosis, models of progressive kidney fibrosis, we demonstrate increased kidney expression of RIPK3. Mice genetically deficient in RIPK3 displayed decreased kidney fibrosis and improved kidney function relative to WT mice when challenged with UUO or AD. In contrast, mice genetically deficient in mixed-lineage kinase domain-like protein (MLKL), a downstream RIPK3 target, were not protected from UUO-induced kidney fibrosis. We demonstrate a pathway by which RIPK3 promotes fibrogenesis through the AKT-dependent activation of ATP citrate lyase (ACL). Genetic or chemical inhibition of RIPK3 suppressed the phosphorylation of AKT and ACL in response to TGF-β1 in fibroblasts. Inhibition of AKT or ACL suppressed TGF-β1–dependent extracellular matrix production and myofibroblast differentiation in fibroblasts. Pharmacological inhibition of ACL suppressed UUO-induced kidney fibrosis. RIPK3 expression was highly regulated in human CKD kidney. In conclusion, we identify a pathway by which RIPK3 promotes kidney fibrosis independently of MLKL-dependent necroptosis as a promising therapeutic target in CKD.
Mitsuru Imamura, Jong-Seok Moon, Kuei-Pin Chung, Kiichi Nakahira, Thangamani Muthukumar, Roman Shingarev, Stefan W. Ryter, Augustine M.K. Choi, Mary E. Choi
Mesenchymal stem cells (MSCs) can give rise to both adipocytes and osteoblasts, but the molecular mechanisms underlying MSC fate determination remain poorly understood. IκB kinase β (IKKβ), a central coordinator of inflammation and immune responses through activation of NF-κB, has been implicated as a critical molecular link between obesity and metabolic disorders. Here, we show that IKKβ can reciprocally regulate adipocyte and osteoblast differentiation of murine and human MSCs through an NF-κB–independent mechanism. IKKβ is a β-catenin kinase that phosphorylates the conserved degron motif of β-catenin to prime it for β-TrCP–mediated ubiquitination and degradation, thereby increasing adipogenesis and inhibiting osteogenesis in MSCs. Animal studies demonstrated that deficiency of IKKβ in BM mesenchymal stromal cells increased bone mass and decreased BM adipocyte formation in adult mice. In humans, IKKβ expression in adipose tissue was also positively associated with increased adiposity and elevated β-catenin phosphorylation. These findings suggest IKKβ as a key molecular switch that regulates MSC fate, and they provide potentially novel mechanistic insights into the understanding of the cross-regulation between the evolutionarily conserved IKKβ and Wnt/β-catenin signaling pathways. The IKKβ-Wnt axis we uncovered may also have important implications for development, homeostasis, and disease pathogenesis.
Yipeng Sui, Zun Liu, Se-Hyung Park, Sean E. Thatcher, Beibei Zhu, Joseph P. Fernandez, Henrik Molina, Philip A. Kern, Changcheng Zhou
The mechanisms underlying the development and natural progression of the airway mucus defect in cystic fibrosis (CF) remain largely unclear. New animal models of CF, coupled with imaging using micro-optical coherence tomography, can lead to insights regarding these questions. The Cftr–/– (KO) rat allows for longitudinal examination of the development and progression of airway mucus abnormalities. The KO rat exhibits decreased periciliary depth, hyperacidic pH, and increased mucus solid content percentage; however, the transport rates and viscoelastic properties of the mucus are unaffected until the KO rat ages. Airway submucosal gland hypertrophy develops in the KO rat by 6 months of age. Only then does it induce increased mucus viscosity, collapse of the periciliary layer, and delayed mucociliary transport; stimulation of gland secretion potentiates this evolution. These findings could be reversed by bicarbonate repletion but not pH correction without counterion donation. These studies demonstrate that abnormal surface epithelium in CF does not cause delayed mucus transport in the absence of functional gland secretions. Furthermore, abnormal bicarbonate transport represents a specific target for restoring mucus clearance, independent of effects on periciliary collapse. Thus, mature airway secretions are required to manifest the CF defect primed by airway dehydration and bicarbonate deficiency.
Susan E. Birket, Joy M. Davis, Courtney M. Fernandez, Katherine L. Tuggle, Ashley M. Oden, Kengyeh K. Chu, Guillermo J. Tearney, Michelle V. Fanucchi, Eric J. Sorscher, Steven M. Rowe
Idiopathic pulmonary fibrosis (IPF) is a fatal disease of unknown etiology characterized by a compositionally and mechanically altered extracellular matrix. Poor understanding of the origin of α-smooth muscle actin (α-SMA) expressing myofibroblasts has hindered curative therapies. Though proposed as a source of myofibroblasts in mammalian tissues, identification of microvascular pericytes (PC) as contributors to α-SMA–expressing populations in human IPF and the mechanisms driving this accumulation remain unexplored. Here, we demonstrate enhanced detection of α-SMA+ cells coexpressing the PC marker neural/glial antigen 2 in the human IPF lung. Isolated human PC cultured on decellularized IPF lung matrices adopt expression of α-SMA, demonstrating that these cells undergo phenotypic transition in response to direct contact with the extracellular matrix (ECM) of the fibrotic human lung. Using potentially novel human lung–conjugated hydrogels with tunable mechanical properties, we decoupled PC responses to matrix composition and stiffness to show that α-SMA+ PC accumulate in a mechanosensitive manner independent of matrix composition. PC activated with TGF-β1 remodel the normal lung matrix, increasing tissue stiffness to facilitate the emergence of α-SMA+ PC via MKL-1/MTRFA mechanotranduction. Nintedanib, a tyrosine-kinase inhibitor approved for IPF treatment, restores the elastic modulus of fibrotic lung matrices to reverse the α-SMA+ phenotype. This work furthers our understanding of the role that microvascular PC play in the evolution of IPF, describes the creation of an ex vivo platform that advances the study of fibrosis, and presents a potentially novel mode of action for a commonly used antifibrotic therapy that has great relevance for human disease.
Parid Sava, Anand Ramanathan, Amelia Dobronyi, Xueyan Peng, Huanxing Sun, Adrian Ledesma-Mendoza, Erica L. Herzog, Anjelica L. Gonzalez
Primary and secondary hypertension are major risk factors for cardiovascular disease, the leading cause of death worldwide. Elevated secretion of aldosterone resulting from primary aldosteronism (PA) is a key driver of secondary hypertension. Here, we report an unexpected role for the ubiquitin ligase Siah1 in adrenal gland development and PA. Siah1a–/– mice exhibit altered adrenal gland morphology, as reflected by a diminished X-zone, enlarged medulla, and dysregulated zonation of the glomerulosa as well as increased aldosterone levels and aldosterone target gene expression and reduced plasma potassium levels. Genes involved in catecholamine biosynthesis and cAMP signaling are upregulated in the adrenal glands of Siah1a–/– mice, while genes related to retinoic acid signaling and cholesterol biosynthesis are downregulated. Loss of Siah1 leads to increased expression of the Siah1 substrate PIAS1, an E3 SUMO protein ligase implicated in the suppression of LXR, a key regulator of cholesterol levels in the adrenal gland. In addition, SIAH1 sequence variants were identified in patients with PA; such variants impaired SIAH1 ubiquitin ligase activity, resulting in elevated PIAS1 expression. These data identify a role for the Siah1-PIAS1 axis in adrenal gland organization and function and point to possible therapeutic targets for hyperaldosteronism.
Marzia Scortegagna, Annabel Berthon, Nikolaos Settas, Andreas Giannakou, Guillermina Garcia, Jian-Liang Li, Brian James, Robert C. Liddington, José G. Vilches-Moure, Constantine A. Stratakis, Ze’ev A. Ronai
Expansion of novel therapeutics to all patients with cystic fibrosis (CF) requires personalized CFTR modulator therapy. We have developed nasospheroids, a primary cell culture–based model derived from individual CF patients and healthy subjects by a minimally invasive nasal biopsy. Confocal microscopy was utilized to measure CFTR activity by analyzing changes in cross-sectional area over time that resulted from CFTR-mediated ion and fluid movement. Both the rate of change over time and AUC were calculated. Non-CF nasospheroids with active CFTR-mediated ion and fluid movement showed a reduction in cross-sectional area, whereas no changes were observed in CF spheroids. Non-CF spheroids treated with CFTR inhibitor lost responsiveness for CFTR activation. However, nasospheroids from F508del CF homozygotes that were treated with lumacaftor and ivacaftor showed a significant reduction in cross-sectional area, indicating pharmacologic rescue of CFTR function. This model employs a simple measurement of size corresponding to changes in CFTR activity and is applicable for detection of small changes in CFTR activity from individual patients in vitro. Advancements of this technique will provide a robust model for individualized prediction of CFTR modulator efficacy.
Jennifer S. Guimbellot, Justin M. Leach, Imron G. Chaudhry, Nancy L. Quinney, Susan E. Boyles, Michael Chua, Inmaculada Aban, Ilona Jaspers, Martina Gentzsch
No posts were found with this tag.