Restoring blood flow after myocardial infarction (MI) is essential for survival of existing and newly regenerated tissue. Endogenous vascular repair processes are deployed following injury but are poorly understood. We sought to determine whether developmental mechanisms of coronary vessel formation are intrinsically reactivated in the adult mouse after MI. Using pulse-chase genetic lineage tracing, we establish that de novo vessel formation constitutes a substantial component of the neovascular response, with apparent cellular contributions from the endocardium and coronary sinus. The adult heart reverts to its former hypertrabeculated state and repeats the process of compaction, which may facilitate endocardium-derived neovascularization. The capacity for angiogenic sprouting of the coronary sinus vein, the adult derivative of the sinus venosus, may also reflect its embryonic origin. The quiescent epicardium is reactivated and, while direct cellular contribution to new vessels is minimal, it supports the directional expansion of the neovessel network toward the infarcted myocardium. Thymosin β4, a peptide with roles in vascular development, was required for endocardial compaction, epicardial vessel expansion, and smooth muscle cell recruitment. Insight into pathways that regulate endogenous vascular repair, drawing on comparisons with development, may reveal novel targets for therapeutically enhancing neovascularization.
Karina N. Dubé, Tonia M. Thomas, Sonali Munshaw, Mala Rohling, Paul R. Riley, Nicola Smart
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
Environmental exposures pose a significant threat to human health. However, it is often difficult to study toxicological mechanisms in human subjects due to ethical concerns. Plant-derived aristolochic acids are among the most potent nephrotoxins and carcinogens discovered to date, yet the mechanism of bioactivation in humans remains poorly understood. Microphysiological systems (organs-on-chips) provide an approach to examining the complex, species-specific toxicological effects of pharmaceutical and environmental chemicals using human cells. We microfluidically linked a kidney-on-a-chip with a liver-on-a-chip to determine the mechanisms of bioactivation and transport of aristolochic acid I (AA-I), an established nephrotoxin and human carcinogen. We demonstrate that human hepatocyte-specific metabolism of AA-I substantially increases its cytotoxicity toward human kidney proximal tubular epithelial cells, including formation of aristolactam adducts and release of kidney injury biomarkers. Hepatic biotransformation of AA-I to a nephrotoxic metabolite involves nitroreduction, followed by sulfate conjugation. Here, we identify, in a human tissue-based system, that the sulfate conjugate of the hepatic NQO1-generated aristolactam product of AA-I (AL-I-NOSO3) is the nephrotoxic form of AA-I. This conjugate can be transported out of liver via MRP membrane transporters and then actively transported into kidney tissue via one or more organic anionic membrane transporters. This integrated microphysiological system provides an ex vivo approach for investigating organ-organ interactions, whereby the metabolism of a drug or other xenobiotic by one tissue may influence its toxicity toward another, and represents an experimental approach for studying chemical toxicity related to environmental and other toxic exposures.
Shih-Yu Chang, Elijah J. Weber, Viktoriya S. Sidorenko, Alenka Chapron, Catherine K. Yeung, Chunying Gao, Qingcheng Mao, Danny Shen, Joanne Wang, Thomas A. Rosenquist, Kathleen G. Dickman, Thomas Neumann, Arthur P. Grollman, Edward J. Kelly, Jonathan Himmelfarb, David L. Eaton
IL-7 regulates homeostatic mechanisms that maintain the overall size of the T cell pool throughout life. We show that, under steady-state conditions, IL-7 signaling is principally mediated by activation of signal transducers and activators of transcription 5 (STAT5). In contrast, under lymphopenic conditions, there is a modulation of STAT1 expression resulting in an IL-7–dependent STAT1 and STAT5 activation. Consequently, the IL-7–induced transcriptome is altered with enrichment of IFN-stimulated genes (ISGs). Moreover, STAT1 overexpression was associated with reduced survival in CD4+ T cells undergoing lymphopenia-induced proliferation (LIP). We propose a model in which T cells undergoing LIP upregulate STAT1 protein, “switching on” an alternate IL-7–dependent program. This mechanism could be a physiological process to regulate the expansion and size of the CD4+ T cell pool. During HIV infection, the virus could exploit this pathway, leading to the homeostatic dysregulation of the T cell pools observed in these patients.
Cecile Le Saout, Megan A. Luckey, Alejandro V. Villarino, Mindy Smith, Rebecca B. Hasley, Timothy G. Myers, Hiromi Imamichi, Jung-Hyun Park, John J. O’Shea, H. Clifford Lane, Marta Catalfamo
Tregs hold great promise as a cellular therapy for multiple immunologically mediated diseases, given their ability to control immune responses. The success of such strategies depends on the expansion of healthy, suppressive Tregs ex vivo and in vivo following the transfer. In clinical studies, levels of transferred Tregs decline sharply in the blood within a few days of the transfer. Tregs have a high rate of apoptosis. Here, we describe a new mechanism of Treg self-inflicted damage. We show that granzymes A and -B (GrA and GrB), which are highly upregulated in human Tregs upon stimulation, leak out of cytotoxic granules to induce cleavage of cytoplasmic and nuclear substrates, precipitating apoptosis in target cells. GrA and GrB substrates were protected from cleavage by inhibiting granzyme activity in vitro. Additionally, we show — by using cytometry by time of flight (CYTOF) — an increase in GrB-expressing Tregs in the peripheral blood and renal allografts of transplant recipients undergoing rejection. These GrB-expressing Tregs showed an activated phenotype but were significantly more apoptotic than non–GrB expressing Tregs. This potentially novel finding improves our understanding of Treg survival and suggests that manipulating Gr expression or activity might be useful for designing more effective Treg therapies.
Esilida Sula Karreci, Siawosh K. Eskandari, Farokh Dotiwala, Sujit K. Routray, Ahmed T. Kurdi, Jean Pierre Assaker, Pavlo Luckyanchykov, Albana B. Mihali, Omar Maarouf, Thiago J. Borges, Abdullah Alkhudhayri, Kruti R. Patel, Amr Radwan, Irene Ghobrial, Martina McGrath, Anil Chandraker, Leonardo V. Riella, Wassim Elyaman, Reza Abdi, Judy Lieberman, Jamil Azzi
The development of a highly effective vaccine remains a key strategic goal to aid the control and eventual eradication of Plasmodium falciparum malaria. In recent years, the reticulocyte-binding protein homolog 5 (RH5) has emerged as the most promising blood-stage P. falciparum candidate antigen to date, capable of conferring protection against stringent challenge in Aotus monkeys. We report on the first clinical trial to our knowledge to assess the RH5 antigen — a dose-escalation phase Ia study in 24 healthy, malaria-naive adult volunteers. We utilized established viral vectors, the replication-deficient chimpanzee adenovirus serotype 63 (ChAd63), and the attenuated orthopoxvirus modified vaccinia virus Ankara (MVA), encoding RH5 from the 3D7 clone of P. falciparum. Vaccines were administered i.m. in a heterologous prime-boost regimen using an 8-week interval and were well tolerated. Vaccine-induced anti-RH5 serum antibodies exhibited cross-strain functional growth inhibition activity (GIA) in vitro, targeted linear and conformational epitopes within RH5, and inhibited key interactions within the RH5 invasion complex. This is the first time to our knowledge that substantial RH5-specific responses have been induced by immunization in humans, with levels greatly exceeding the serum antibody responses observed in African adults following years of natural malaria exposure. These data support the progression of RH5-based vaccines to human efficacy testing.
Ruth O. Payne, Sarah E. Silk, Sean C. Elias, Kazutoyo Miura, Ababacar Diouf, Francis Galaway, Hans de Graaf, Nathan J. Brendish, Ian D. Poulton, Oliver J. Griffiths, Nick J. Edwards, Jing Jin, Geneviève M. Labbé, Daniel G.W. Alanine, Loredana Siani, Stefania Di Marco, Rachel Roberts, Nicky Green, Eleanor Berrie, Andrew S. Ishizuka, Carolyn M. Nielsen, Martino Bardelli, Frederica D. Partey, Michael F. Ofori, Lea Barfod, Juliana Wambua, Linda M. Murungi, Faith H. Osier, Sumi Biswas, James S. McCarthy, Angela M. Minassian, Rebecca Ashfield, Nicola K. Viebig, Fay L. Nugent, Alexander D. Douglas, Johan Vekemans, Gavin J. Wright, Saul N. Faust, Adrian V.S. Hill, Carole A. Long, Alison M. Lawrie, Simon J. Draper
Phospholipase A2 (PLA2) enzymes regulate the formation of eicosanoids and lysophospholipids that contribute to allergic airway inflammation. Secreted PLA2 group X (sPLA2-X) was recently found to be increased in the airways of asthmatics and is highly expressed in airway epithelial cells and macrophages. In the current study, we show that allergen exposure increases sPLA2-X in humans and in mice, and that global deletion of Pla2g10 results in a marked reduction in airway hyperresponsiveness (AHR), eosinophil and T cell trafficking to the airways, airway occlusion, generation of type-2 cytokines by antigen-stimulated leukocytes, and antigen-specific immunoglobulins. Further, we found that Pla2g10–/– mice had reduced IL-33 levels in BALF, fewer type-2 innate lymphoid cells (ILC2s) in the lung, less IL-33–induced IL-13 expression in mast cells, and a marked reduction in both the number of newly recruited macrophages and the M2 polarization of these macrophages in the lung. These results indicate that sPLA2-X serves as a central regulator of both innate and adaptive immune response to proteolytic allergen.
James D. Nolin, Ying Lai, Herbert Luke Ogden, Anne M. Manicone, Ryan C. Murphy, Dowon An, Charles W. Frevert, Farideh Ghomashchi, Gajendra S. Naika, Michael H. Gelb, Gail M. Gauvreau, Adrian M. Piliponsky, William A. Altemeier, Teal S. Hallstrand
Maternal obesity is a global health problem that increases offspring obesity risk. The metabolic pathways underlying early developmental programming in human infants at risk for obesity remain poorly understood, largely due to barriers in fetal/infant tissue sampling. Utilizing umbilical cord–derived mesenchymal stem cells (uMSC) from offspring of normal weight and obese mothers, we tested whether energy metabolism and gene expression differ in differentiating uMSC myocytes and adipocytes, in relation to maternal obesity exposures and/or neonatal adiposity. Biomarkers of incomplete β-oxidation were uniquely positively correlated with infant adiposity and maternal lipid levels in uMSC myocytes from offspring of obese mothers only. Metabolic and biosynthetic processes were enriched in differential gene expression analysis related to maternal obesity. In uMSC adipocytes, maternal obesity and lipids were associated with downregulation in multiple insulin-dependent energy-sensing pathways including PI3K and AMPK. Maternal lipids correlated with uMSC adipocyte upregulation of the mitochondrial respiratory chain but downregulation of mitochondrial biogenesis. Overall, our data revealed cell-specific alterations in metabolism and gene expression that correlated with maternal obesity and adiposity of their offspring, suggesting tissue-specific metabolic and regulatory changes in these newborn cells. We provide important insight into potential developmental programming mechanisms of increased obesity risk in offspring of obese mothers.
Peter R. Baker II, Zachary Patinkin, Allison L.B. Shapiro, Becky A. De La Houssaye, Michael Woontner, Kristen E. Boyle, Lauren Vanderlinden, Dana Dabelea, Jacob E. Friedman
Neurogenic heterotopic ossification (NHO) is the formation of ectopic bone generally in muscles surrounding joints following spinal cord or brain injury. We investigated the mechanisms of NHO formation in 64 patients and a mouse model of spinal cord injury–induced NHO. We show that marrow from human NHOs contains hematopoietic stem cell (HSC) niches, in which mesenchymal stromal cells (MSCs) and endothelial cells provide an environment supporting HSC maintenance, proliferation, and differentiation. The transcriptomic signature of MSCs from NHOs shows a neuronal imprinting associated with a molecular network required for HSC support. We demonstrate that oncostatin M (OSM) produced by activated macrophages promotes osteoblastic differentiation and mineralization of human muscle-derived stromal cells surrounding NHOs. The key role of OSM was confirmed using an experimental model of NHO in mice defective for the OSM receptor (OSMR). Our results provide strong evidence that macrophages contribute to NHO formation through the osteogenic action of OSM on muscle cells within an inflammatory context and suggest that OSM/OSMR could be a suitable therapeutic target. Altogether, the evidence of HSCs in ectopic bones growing at the expense of soft tissue in spinal cord/brain-injured patients indicates that inflammation and muscle contribute to HSC regulation by the brain-bone-blood triad.
Frédéric Torossian, Bernadette Guerton, Adrienne Anginot, Kylie A. Alexander, Christophe Desterke, Sabrina Soave, Hsu-Wen Tseng, Nassim Arouche, Laetitia Boutin, Irina Kulina, Marjorie Salga, Beulah Jose, Allison R. Pettit, Denis Clay, Nathalie Rochet, Erica Vlachos, Guillaume Genet, Charlotte Debaud, Philippe Denormandie, François Genet, Natalie A. Sims, Sébastien Banzet, Jean-Pierre Levesque, Jean-Jacques Lataillade, Marie-Caroline Le Bousse-Kerdilès
Accumulation of lipid droplets and inflammatory cell infiltration is the hallmark of nonalcoholic steatohepatitis (NASH). The roles of noncoding RNAs in NASH are less known. We aim to elucidate the function of miR-141/200c in diet-induced NASH. WT and miR-141/200c–/– mice were fed a methionine and choline deficient (MCD) diet for 2 weeks to assess markers of steatosis, liver injury, and inflammation. Hepatic miR-141 and miR-200c RNA levels were highly induced in human patients with NASH fatty liver and in WT MCD mice. miR-141/200c–/– MCD mice had reduced liver weights and triglyceride (TG) levels, which was associated with increased microsomal TG transfer protein (MTTP) and PPARα but reduced SREBP1c and FAS expression. Inflammation was attenuated and F4/80 macrophage activation was suppressed in miR-141/200c–/– mice, as evidenced by decreased serum aminotransferases and IL-6 and reduced hepatic proinflammatory, neutrophil, and profibrotic genes. Treatment with LPS in BM-derived macrophages isolated from miR-200c/141–/– mice polarized macrophages toward the M2 antiinflammatory state by increasing Arg1 and IL-10 levels while decreasing the M1 marker iNOS. In addition, elevated phosphorylated AMPK (p-AMPK), p-AKT, and p-GSK3β and diminished TLR4 and p-mTOR/p-4EBP1 proteins were observed. Lipidomics and metabolomics revealed alterations of TG and phosphatidylcholine (PC) lipid species by miR-141/200c deficiency. In summary, miR-141/200c deficiency diminished NASH-associated hepatic steatosis and inflammation by reprogramming lipid and inflammation signaling pathways.
Melanie Tran, Sang-Min Lee, Dong-Ju Shin, Li Wang
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