Capillary malformation-arteriovenous malformation (CM-AVM) is a blood vascular anomaly caused by inherited loss of function mutations in RASA1 or EPHB4 genes that encode p120 Ras GTPase-activating protein (p120 RasGAP/RASA1) and Ephrin receptor B4 (EPHB4) respectively. However, whether RASA1 and EPHB4 function in the same molecular signaling pathway to regulate the blood vasculature is uncertain. Here, we show that induced endothelial cell (EC)-specific disruption of Ephb4 in mice results in accumulation of collagen IV in the EC endoplasmic reticulum leading to EC apoptotic death and defective developmental, neonatal and pathological angiogenesis, as reported previously in induced EC-specific RASA1-deficient mice. Moreover, defects in angiogenic responses in EPHB4-deficient mice can be rescued by drugs that inhibit signaling through the Ras pathway and drugs that promote collagen IV export from the ER. However, EPHB4 mutant mice that express a form of EPHB4 that is unable to physically engage RASA1 but retains protein tyrosine kinase activity show normal angiogenic responses. These findings provide strong evidence that RASA1 and EPHB4 function in the same signaling pathway to protect against the development of CM-AVM independent of physical interaction and have important implications with regards possible means of treatment of this disease.
Di Chen, Elizabeth D. Hughes, Thomas L. Saunders, Jiangping Wu, Magda N. Hernández Vásquez, Taija Makinen, Philip D. King
The G-protein coupled C-X-C chemokine receptor 4 (CXCR4) is a candidate therapeutic target for tissue fibrosis. A novel fully human single-domain antibody-like scaffold i-body AD-114-PA600 (AD-114) with specific high binding affinity to CXCR4 has been developed. To define the renoprotective role, AD-114 was administrated in a mouse model of renal fibrosis induced by folic acid (FA). Increased extracellular matrix (ECM) accumulation, macrophage infiltration, inflammatory response, transforming growth factor beta-1 (TGF-β1) expression and fibroblasts activation were observed in kidneys of mice with FA-induced nephropathy. These markers were normalized or partially reversed by AD-114 treatment. In vitro studies demonstrated AD-114 blocked TGF-β1-induced upregulated expression of ECM, matrix metallopeptidase-2 (MMP-2) and downstream p38 mitogen-activated protein kinases (p38 MAPK) and Phosphoinositide 3-kinases (PI3K)/AKT/ mammalian target of rapamycin (mTOR) signaling pathways in a renal proximal tubular cell line. Additionally, these renoprotective effects were validated in a second model of unilateral ureteral obstruction (UUO) using a second generation of AD-114 (Fc-fused AD-114, also named AD-214). Collectively, these results suggest a renoprotective role of AD-114 as it inhibited the chemotactic function of CXCR4 as well as blocked CXCR4 downstream p38 MAPK and PI3K/AKT/mTOR signaling, which establish a therapeutic strategy for AD-114 targeting CXCR4 to limit renal fibrosis.
Qinghua Cao, Chunling Huang, Hao Yi, Anthony J. Gill, Angela Chou, Michael Foley, Chris G. Hosking, Kevin K. Lim, Cristina F. Triffon, Ying Shi, Xin-Ming Chen, Carol A. Pollock
Increased adipose tissue macrophages (ATM) correlate with metabolic dysfunction in humans and are causal in development of insulin resistance in mice. Recent bulk and single cell transcriptomics studies reveal a wide spectrum of gene expression signatures possible for macrophages that depends on context, but the signatures of human ATM subtypes are not well defined in obesity and diabetes. We profiled three prominent ATM subtypes from human adipose tissue in obesity and determined their relationship to type 2 diabetes. Visceral (VAT) and subcutaneous (SAT) adipose tissue samples were collected from diabetic and non-diabetic obese subjects to evaluate cellular content and gene expression. VAT CD206+CD11c− ATMs were increased in diabetic subjects, scavenger receptor-rich with low intracellular lipids, secreted proinflammatory cytokines, and diverged significantly from two CD11c+ ATM subtypes, which were lipid-laden, lipid antigen presenting, and overlapped with monocyte signatures. Furthermore, diabetic VAT was enriched for CD206+CD11c− ATM and inflammatory signatures, scavenger receptors, and MHC II antigen presentation genes. VAT immunostaining found CD206+CD11c− ATMs concentrated in vascularized lymphoid clusters adjacent to CD206−CD11c+ ATMs, while CD206+CD11c+ were distributed between adipocytes. Our results suggest ATM subtype-specific profiles that uniquely contribute to the phenotypic variation in obesity.
Lindsey A. Muir, Kae Won Cho, Lynn M. Geletka, Nicki A. Baker, Carmen G. Flesher, Anne P. Ehlers, Niko Kaciroti, Stephen Lindsly, Scott Ronquist, Indika Rajapakse, Robert W. O’Rourke, Carey N. Lumeng
Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an arrhythmia syndrome due to gene mutations that render RYR2 calcium release channels hyperactive, causing spontaneous calcium release and delayed afterdepolarizations (DADs). What remains unknown is the cellular source of ventricular arrhythmia triggered by DADs – Purkinje cells in the conduction system or ventricular cardiomyocytes in the working myocardium. To answer this question, we used a genetic approach in mice to knock out cardiac calsequestrin either in Purkinje cells or in ventricular cardiomyocytes. Total loss of calsequestrin in the heart causes a severe CPVT phenotype in mice and humans. We found that loss of calsequestrin only in ventricular myocytes produced a full-blown CPVT phenotype, whereas mice with loss of calsequestrin only in Purkinje cells were comparable to wild-type mice. Subendocardial chemical ablation or restoration of calsequestrin expression in subendocardial cardiomyocytes neighboring Purkinje cells was sufficient to protect against catecholamine-induced arrhythmias. In silico modeling demonstrated that DADs in ventricular myocardium can trigger full action potentials in the Purkinje fiber, but not vice versa. Hence, ectopic beats in CPVT are likely generated at the Purkinje-myocardial junction via a heretofore unrecognized tissue mechanism, whereby DADs in the ventricular myocardium trigger full action potentials in adjacent Purkinje cells.
Daniel J. Blackwell, Michela Faggioni, Matthew J. Wleklinski, Nieves Gomez-Hurtado, Raghav Venkataraman, Chelsea E. Gibbs, Franz J. Baudenbacher, Shiaoching Gong, Glenn I. Fishman, Patrick M. Boyle, Karl Pfeifer, Bjorn C. Knollmann
BACKGROUND. Adenoviral (Ad)-vectored vaccines are typically administered via intramuscular injection to humans, incapable of inducing respiratory mucosal immunity. However, aerosol delivery of Ad-vectored vaccines remains poorly characterized and its ability to induce mucosal immunity in humans is unknown. This phase 1b trial was to evaluate the safety and immunogenicity of human serotype-5 Ad-vectored tuberculosis (TB) vaccine (AdHu5Ag85A) delivered to humans via inhaled aerosol or intramuscular injection. METHODS. 31 healthy, previously BCG-vaccinated adults were enrolled. AdHu5Ag85A was administered by single-dose aerosol using Aeroneb® Solo Nebulizer or by intramuscular (IM) injection. The study consisted of the low dose (LD) aerosol, high dose (HD) aerosol and IM groups. The adverse events were assessed at various times post-vaccination. Immunogenicity data were collected from the peripheral blood and bronchoalveolar lavage samples at baseline and select timepoints post-vaccination. RESULTS. The nebulized aerosol droplets were <5.39µm in size. Both LD and HD of AdHu5Ag85A administered by aerosol inhalation and IM injection were safe and well-tolerated. Both aerosol doses, particularly LD, but not IM, vaccination markedly induced airway tissue-resident memory CD4 and CD8 T cells of polyfunctionality. While as expected, IM vaccination induced Ag85A-specific T cell responses in the blood, the LD aerosol vaccination also elicited such T cells in the blood. Furthermore, the LD aerosol vaccination induced persisting transcriptional changes in alveolar macrophages. CONCLUSIONS. Inhaled aerosol delivery of Ad-vectored vaccine is a safe and superior way to elicit respiratory mucosal immunity. This study warrants further development of aerosol vaccine strategies against respiratory pathogens including TB and COVID-19. TRIAL REGISTRATION. This trial is registered with ClinicalTrial.gov, NCT# 02337270. FUNDING. The Canadian Institutes for Health Research and the Natural Sciences and Engineering Research Council of Canada.
Mangalakumari Jeyanathan, Dominik K. Fritz, Sam Afkhami, Emilio Aguirre, Karen J. Howie, Anna Zganiacz, Anna Dvorkin-Gheva, Michael R. Thompson, Richard Silver, Ruth P. Cusack, Brian D. Lichty, Paul M. O'Byrne, Martin Kolb, Maria Fe C. Medina, Myrna B. Dolovich, Imran Satia, Gail M Gauvreau, Zhou Xing, Fiona Smaill
Short stature is a major skeletal phenotype in osteogenesis imperfecta (OI), a genetic disorder mainly caused by mutations in genes encoding type I collagen. However, the underlying mechanism is poorly understood and no effective treatment is available. In OI mice that carry a G610C mutation in COL1A2, we previously found that mature hypertrophic chondrocytes (HCs) are exposed to cell stress due to accumulation of misfolded mutant type I procollagen in the endoplasmic reticulum (ER). By fate mapping analysis of HCs in G610C OI mice, we found that HCs stagnate in the growth plate, inhibiting translocation of HC descendants to the trabecular area and their differentiation to osteoblasts. Treatment with 4-phenylbutyric acid (4PBA), a chemical chaperone, restored HC ER structure and rescued this inhibition, resulting in enhanced longitudinal bone growth in G610C OI mice. Interestingly, the effects of 4PBA on ER dilation were limited in osteoblasts and the bone fragility was not ameliorated. These results highlight the importance of targeting HCs to treat growth deficiency in OI. Our findings demonstrate that HC dysfunction induced by ER disruption plays a critical role in the pathogenesis of OI growth deficiency, which lays the foundation for developing new therapies for OI.
Amanda L. Scheiber, Kevin J. Wilkinson, Akiko Suzuki, Motomi Enomoto-Iwamoto, Takashi Kaito, Kathryn S.E. Cheah, Masahiro Iwamoto, Sergey Leikin, Satoru Otsuru
Cytomegalovirus (CMV) infection remains an important cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Several investigators have reported that adaptive NKG2C+ NK cells persistently expand during CMV reactivation. In our study, two cohorts were enrolled to explored the relationships among the NKG2C genotype, NKG2C+ NK cell reconstitution, and CMV infection. Multivariate analysis showed that donor NKG2C gene deletion was an independent prognostic factor for CMV reactivation and refractory CMV reactivation. Furthermore, the quantitative, qualitative reconstitution and anti-CMV function of adaptive NKG2C+ NK cells after transplantation was significantly lower in patients grafted with NKG2Cwt/del donor cells than in those grafted with NKG2Cwt/wt donor cells. The quantitative reconstitution of NKG2C+ NK cells at day 30 after transplantation was significantly lower in patients with treatment-refractory CMV reactivation than in those in the no-CMV-reactivation and CMV-reactivation groups. In humanized CMV-infected mice, we found that compared with those from NKG2Cwt/del donors, adaptive NKG2C+ NK cells from NKG2Cwt/wt donors induced earlier and stronger expansion of NKG2C+ NK cells and earlier and stronger CMV clearance in vivo. In conclusion, donor NKG2C homozygosity contributes to CMV clearance by promoting the quantitative and qualitative reconstruction of adaptive NKG2C+ NK cells after haploidentical allo-HSCT.
Xing-Xing Yu, Qian-Nan Shang, Xue-Fei Liu, Mei He, Xu-Ying Pei, Xiao-Dong Mo, Meng Lv, Ting-Ting Han, Ming-Rui Huo, Xiaosu Zhao, Ying-Jun Chang, Yu Wang, Xiao-Hui Zhang, Lan-Ping Xu, Kai-Yan Liu, Xiangyu Zhao, Xiaojun Huang
Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal disease characterized by collagen deposition within the lung interstitium. Bacterial infection is associated with increased morbidity and more rapid mortality in IPF patient populations and pathogens such as methicillin-resistant Staphylococcus aureus (MRSA) are commonly isolated from the lungs of hospitalized IPF patients. Despite this, the effects of fibrotic lung injury on critical immune responses to infection remain unknown. In the present study, we show that, like human IPF, fibrotic mice infected with MRSA exhibit increased morbidity and mortality compared to uninfected fibrotic mice. We determine that fibrosis confers a defect in MRSA clearance compared to non-fibrotic mice, resulting from blunted innate immune responses. We show that fibrosis inhibits neutrophil intracellular killing of MRSA through impaired neutrophil elastase (NE) release and oxidative radical production. Additionally, we demonstrate that lung macrophages from fibrotic mice have impaired phagocytosis of MRSA. Our study describes potentially novel impairments to antimicrobial responses upon the development of pulmonary fibrosis and our findings suggest a possible mechanism for why IPF patients are at greater risk of morbidity and mortality related to infection.
Helen I. Warheit-Niemi, Summer J. Edwards, Shuvasree SenGupta, Carole A. Parent, Xiaofeng Zhou, David N. O'Dwyer, Bethany B. Moore
The discovery of the oncometabolite 2-hydroxyglutarate in isocitrate dehydrogenase (IDH)1-mutated tumor entities affirmed the role of metabolism in cancer. Yet, large databases with tissue metabolites that are modulated by IDH1 mutation remain an area of development. Here, we present an unprecedented and valuable resource for tissue metabolites in diffuse glioma and their modulations by IDH1 mutation, histology and tumor treatments in 101 tissue samples from 73 diffuse glioma patients (24 astrocytoma, 17 oligodendroglioma, 32 glioblastoma), investigated by NMR-based metabolomics and supported by RNA sequencing. We discovered comparison-specific metabolites and pathways modulated by IDH1 (“IDH1 mutation status cohort”) and tumor entity. The “Longitudinal investigation cohort” provides metabolic profiles of untreated and corresponding treated glioma samples at first progression. Most interestingly, univariate and multivariate cox regressions and Kaplan Meier analyses revealed that tissue metabolites correlate with progression-free and overall survival. Thus, this study introduces novel candidate prognostic and surrogate metabolite biomarkers for future prospective clinical studies aiming at further refining patient stratification in diffuse glioma. Furthermore, our data will facilitate the generation of so far unanticipated hypotheses for experimental studies to advance our molecular understanding of glioma biology.
Christoph Trautwein, Laimdota Zizmare, Irina Mäurer, Benjamin Bender, Björn Bayer, Ulrike Ernemann, Marcos Tatagiba, Stefan J. Grau, Bernd J. Pichler, Marco Skardelly, Ghazaleh Tabatabai
Discoidin domain receptor 1 (DDR1), a receptor tyrosine kinase activated by collagen, contributes to chronic kidney disease (CKD). However, its role in acute kidney injury (AKI) and subsequent development of kidney fibrosis is not clear. Thus, we performed a model of severe ischemia-reperfusion-induced AKI that progresses to kidney fibrosis in wild-type and Ddr1-null mice. We show that Ddr1-null mice had reduced acute tubular injury, inflammation, and tubulointerstitial fibrosis with overall decreased renal monocyte chemoattractant protein (MCP-1) levels and STAT3 activation. We identified breakpoint cluster region (BCR) protein as a phosphorylated target of DDR1 that controls MCP-1 production in renal proximal tubule epithelial cells. DDR1-induced BCR phosphorylation or BCR downregulation increased MCP-1 secretion, suggesting that BCR negatively regulates the levels of MCP-1. Mechanistically, phosphorylation or downregulation of BCR increases β-catenin activity and in turn MCP-1 production. Finally, we show that DDR1-mediated STAT3 activation is required to stimulate the secretion of TGF-β. Thus, DDR1 contributes to acute and chronic kidney injury by regulating BCR and STAT3 phosphorylation and in turn the production of MCP-1 and TGF-β. These findings identify DDR1 an attractive therapeutic target for ameliorating both pro-inflammatory and pro-fibrotic signaling in kidney disease.
Corina M. Borza, Gema Bolas, Fabian Bock, Xiuqi Zhang, Favour C. Akabogu, Ming-Zhi Zhang, Mark de Caestecker, Min Yang, Hai-chun Yang, Ethan Lee, Leslie Gewin, Agnes B. Fogo, W. Hayes McDonald, Roy Zent, Ambra Pozzi
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