Person: Wagers, Amy
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Wagers
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Amy
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Wagers, Amy
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Publication Overexpressing IRS1 in Endothelial Cells Enhances Angioblast Differentiation and Wound Healing in Diabetes and Insulin Resistance(American Diabetes Association, 2016) Katagiri, Sayaka; Park, Kyoungmin; Maeda, Yasutaka; Rao, Tata Nageswara; Khamaisi, Mogher; Li, Qian; Yokomizo, Hisashi; Mima, Akira; Lancerotto, Luca; Wagers, Amy; Orgill, Dennis; King, GeorgeThe effect of enhancing insulin’s actions in endothelial cells (ECs) to improve angiogenesis and wound healing was studied in obesity and diabetes. Insulin receptor substrate 1 (IRS1) was overexpressed in ECs using the VE-cadherin promoter to create ECIRS1 TG mice, which elevated pAkt activation and expressions of vascular endothelial growth factor (VEGF), Flk1, and VE-cadherin in ECs and granulation tissues (GTs) of full-thickness wounds. Open wound and epithelialization rates and angiogenesis significantly improved in normal mice and high fat (HF) diet–induced diabetic mice with hyperinsulinemia in ECIRS1 TG versus wild type (WT), but not in insulin-deficient diabetic mice. Increased angioblasts and EC numbers in GT of ECIRS1 mice were due to proliferation in situ rather than uptake. GT in HF-fed diabetic mice exhibited parallel decreases in insulin and VEGF-induced pAkt and EC numbers by >50% without changes in angioblasts versus WT mice, which were improved in ECIRS1 TG mice on normal chow or HF diet. Thus, HF-induced diabetes impaired angiogenesis by inhibiting insulin signaling in GT to decrease the differentiation of angioblasts to EC, which was normalized by enhancing insulin’s action targeted to EC, a potential target to improve wound healing in diabetes and obesity.Publication Direct Reprogramming of Mouse Fibroblasts into Functional Skeletal Muscle Progenitors(Elsevier, 2018) Bar-Nur, Ori; Gerli, Mattia F.M.; Di Stefano, Bruno; Almada, Albert; Galvin, Amy; Coffey, Amy; Huebner, Aaron; Feige, Peter; Verheul, Cassandra; Cheung, Priscilla; Payzin-Dogru, Duygu; Paisant, Sylvain; Anselmo, Anthony; Sadreyev, Ruslan; Ott, Harald; Tajbakhsh, Shahragim; Rudnicki, Michael A.; Wagers, Amy; Hochedlinger, KonradSummary Skeletal muscle harbors quiescent stem cells termed satellite cells and proliferative progenitors termed myoblasts, which play pivotal roles during muscle regeneration. However, current technology does not allow permanent capture of these cell populations in vitro. Here, we show that ectopic expression of the myogenic transcription factor MyoD, combined with exposure to small molecules, reprograms mouse fibroblasts into expandable induced myogenic progenitor cells (iMPCs). iMPCs express key skeletal muscle stem and progenitor cell markers including Pax7 and Myf5 and give rise to dystrophin-expressing myofibers upon transplantation in vivo. Notably, a subset of transplanted iMPCs maintain Pax7 expression and sustain serial regenerative responses. Similar to satellite cells, iMPCs originate from Pax7+ cells and require Pax7 itself for maintenance. Finally, we show that myogenic progenitor cell lines can be established from muscle tissue following small-molecule exposure alone. This study thus reports on a robust approach to derive expandable myogenic stem/progenitor-like cells from multiple cell types.Publication Cell Type of Origin Influences the Molecular and Functional Properties of Mouse Induced Pluripotent Stem Cells(Nature Publishing Group, 2010) Polo, Jose M.; Liu, Susanna; Figueroa, Maria Eugenia; Kulalert, Warakorn; Eminli, Sarah; Tan, Kah Yong; Apostolou, Effie; Stadtfeld, Matthias; Li, Yushan; Shioda, Toshihiro; Natesan, Sridaran; Wagers, Amy; Melnick, Ari; Evans, Todd; Hochedlinger, KonradInduced pluripotent stem cells (iPSCs) have been derived from various somatic cell populations through ectopic expression of defined factors. It remains unclear whether iPSCs generated from different cell types are molecularly and functionally similar. Here we show that iPSCs obtained from mouse fibroblasts, hematopoietic and myogenic cells exhibit distinct transcriptional and epigenetic patterns. Moreover, we demonstrate that cellular origin influences the in vitro differentiation potentials of iPSCs into embryoid bodies and different hematopoietic cell types. Notably, continuous passaging of iPSCs largely attenuates these differences. Our results suggest that early-passage iPSCs retain a transient epigenetic memory of their somatic cells of origin, which manifests as differential gene expression and altered differentiation capacity. These observations may influence ongoing attempts to use iPSCs for disease modeling and could also be exploited in potential therapeutic applications to enhance differentiation into desired cell lineages.Publication Cell-Cycle Dependent Expression of a Translocation-Mediated Fusion Oncogene Mediates Checkpoint Adaptation in Rhabdomyosarcoma(Public Library of Science, 2014) Kikuchi, Ken; Hettmer, Simone; Aslam, M. Imran; Michalek, Joel E.; Laub, Wolfram; Wilky, Breelyn A.; Loeb, David M.; Rubin, Brian P.; Wagers, Amy; Keller, CharlesRhabdomyosarcoma is the most commonly occurring soft-tissue sarcoma in childhood. Most rhabdomyosarcoma falls into one of two biologically distinct subgroups represented by alveolar or embryonal histology. The alveolar subtype harbors a translocation-mediated PAX3:FOXO1A fusion gene and has an extremely poor prognosis. However, tumor cells have heterogeneous expression for the fusion gene. Using a conditional genetic mouse model as well as human tumor cell lines, we show that that Pax3:Foxo1a expression is enriched in G2 and triggers a transcriptional program conducive to checkpoint adaptation under stress conditions such as irradiation in vitro and in vivo. Pax3:Foxo1a also tolerizes tumor cells to clinically-established chemotherapy agents and emerging molecularly-targeted agents. Thus, the surprisingly dynamic regulation of the Pax3:Foxo1a locus is a paradigm that has important implications for the way in which oncogenes are modeled in cancer cells.Publication Engineering Escherichia coli into a Protein Delivery System for Mammalian Cells(American Chemical Society, 2015) Reeves, Analise Z.; Spears, William E.; Du, Juan; Tan, Kah Yong; Wagers, Amy; Lesser, CammieMany Gram-negative pathogens encode type 3 secretion systems, sophisticated nanomachines that deliver proteins directly into the cytoplasm of mammalian cells. These systems present attractive opportunities for therapeutic protein delivery applications; however, their utility has been limited by their inherent pathogenicity. Here, we report the reengineering of a laboratory strain of Escherichia coli with a tunable type 3 secretion system that can efficiently deliver heterologous proteins into mammalian cells, thereby circumventing the need for virulence attenuation. We first introduced a 31 kB region of Shigella flexneri DNA that encodes all of the information needed to form the secretion nanomachine onto a plasmid that can be directly propagated within E. coli or integrated into the E. coli chromosome. To provide flexible control over type 3 secretion and protein delivery, we generated plasmids expressing master regulators of the type 3 system from either constitutive or inducible promoters. We then constructed a Gateway-compatible plasmid library of type 3 secretion sequences to enable rapid screening and identification of sequences that do not perturb function when fused to heterologous protein substrates and optimized their delivery into mammalian cells. Combining these elements, we found that coordinated expression of the type 3 secretion system and modified target protein substrates produces a nonpathogenic strain that expresses, secretes, and delivers heterologous proteins into mammalian cells. This reengineered system thus provides a highly flexible protein delivery platform with potential for future therapeutic applications.Publication Growth Differentiation Factor 11 Is a Circulating Factor that Reverses Age-Related Cardiac Hypertrophy(Elsevier BV, 2013) Loffredo, F; Steinhauser, Matthew; Jay, Steven M.; Gannon, Joseph; Pancoast, James R.; Yalamanchi, Pratyusha; Sinha, Manisha; Dall’Osso, Claudia; Khong, Danika; Shadrach, Jennifer; Miller, Christine; Singer, Britta S.; Stewart, Alex; Psychogios, Nikolaos; Gerszten, Robert; Hartigan, Adam J.; Kim, Mi-Jeong; Serwold, Thomas; Wagers, Amy; Lee, RichardThe most common form of heart failure occurs with normal systolic function and often involves cardiac hypertrophy in the elderly. To clarify the biological mechanisms that drive cardiac hypertrophy in aging, we tested the influence of circulating factors using heterochronic parabiosis, a surgical technique in which joining of animals of different ages leads to a shared circulation. After 4 weeks of exposure to the circulation of young mice, cardiac hypertrophy in old mice dramatically regressed, accompanied by reduced cardiomyocyte size and molecular remodeling. Reversal of age-related hypertrophy was not attributable to hemodynamic or behavioral effects of parabiosis, implicating a blood-borne factor. Using modified aptamer-based proteomics, we identified the TGF-b superfamily member GDF11 as a circulating factor in young mice that declines with age. Treatment of old mice to restore GDF11 to youthful levels recapitulated the effects of parabiosis and reversed age-related hypertrophy, revealing a therapeutic opportunity for cardiac aging.Publication Functional genomic screening reveals asparagine dependence as a metabolic vulnerability in sarcoma(eLife Sciences Publications, Ltd, 2015) Hettmer, Simone; Schinzel, Anna C; Tchessalova, Daria; Schneider, Michaela; Parker, Christina L; Bronson, Roderick T; Richards, Nigel GJ; Hahn, William; Wagers, AmyCurrent therapies for sarcomas are often inadequate. This study sought to identify actionable gene targets by selective targeting of the molecular networks that support sarcoma cell proliferation. Silencing of asparagine synthetase (ASNS), an amidotransferase that converts aspartate into asparagine, produced the strongest inhibitory effect on sarcoma growth in a functional genomic screen of mouse sarcomas generated by oncogenic Kras and disruption of Cdkn2a. ASNS silencing in mouse and human sarcoma cell lines reduced the percentage of S phase cells and impeded new polypeptide synthesis. These effects of ASNS silencing were reversed by exogenous supplementation with asparagine. Also, asparagine depletion via the ASNS inhibitor amino sulfoximine 5 (AS5) or asparaginase inhibited mouse and human sarcoma growth in vitro, and genetic silencing of ASNS in mouse sarcoma cells combined with depletion of plasma asparagine inhibited tumor growth in vivo. Asparagine reliance of sarcoma cells may represent a metabolic vulnerability with potential anti-sarcoma therapeutic value. DOI: http://dx.doi.org/10.7554/eLife.09436.001Publication Modified mRNA directs the fate of heart progenitor cells and induces vascular regeneration after myocardial infarction(Nature Publishing Group, 2013) Zangi, Lior; Lui, Kathy O; von Gise, Alexander; Ma, Qing; Ebina, Wataru; Ptaszek, Leon; Später, Daniela; Xu, Huansheng; Tabebordbar, M; Gorbatov, Rostic; Sena, Brena; Nahrendorf, Matthias; Briscoe, David; Li, Ronald A; Wagers, Amy; Rossi, Derrick; Pu, William; Chien, Kenneth RIn a cell-free approach to regenerative therapeutics, transient application of paracrine factors in vivo could be used to alter the behavior and fate of progenitor cells to achieve sustained clinical benefits. Here we show that intramyocardial injection of synthetic modified RNA (modRNA) encoding human vascular endothelial growth factor-A (VEGF-A) results in the expansion and directed differentiation of endogenous heart progenitors in a mouse myocardial infarction model. VEGF-A modRNA markedly improved heart function and enhanced long-term survival of recipients. This improvement was in part due to mobilization of epicardial progenitor cells and redirection of their differentiation toward cardiovascular cell types. Direct in vivo comparison with DNA vectors and temporal control with VEGF inhibitors revealed the greatly increased efficacy of pulse-like delivery of VEGF-A. Our results suggest that modRNA is a versatile approach for expressing paracrine factors as cell fate switches to control progenitor cell fate and thereby enhance long-term organ repair.Publication Use of the parabiotic model in studies of cutaneous wound healing to define the participation of circulating cells(Wiley-Blackwell, 2010) Song, Guodong; Nguyen, Dinh T.; Pietramaggiori, Giorgio; Scherer, Saja; Chen, Bing; Zhan, Qian; Ogawa, Rei; Yannas, I.V.; Wagers, Amy; Orgill, Dennis; Murphy, GeorgePrevious experimental studies to assess the contribution of blood-borne circulating (BBC) cells to cutaneous wound healing have relied on discontinuous pulsing of labeled BBC elements or bone marrow transplant protocols. Such approaches do not allow the examination of stable BBC cells that have matured in a physiologically normal host. We have used a parabiotic murine model for cutaneous wound healing to evaluate the relative contribution of stable populations of peripheral blood cells expressing the green fluorescent protein (GFP) transgene in otherwise normal animals. Circulating cells (mature and immature) expressing the GFP transgene were easily detected and quantified in wounds of GFP− parabiotic twins during all evaluated stages of the healing response. Using multiple antibody probes, the relative contribution of various subsets of BBC cells could be comparatively assessed. In early wounds, some cells expressing mesenchymal epitopes were documented to be of hematopoietic origin, indicating the utility of this model in assessing cell plasticity in the context of tissue regeneration and repair. Application of this approach enables further investigation into the contribution of peripheral blood in normal and abnormal healing responses.Publication Improved Cutaneous Healing in Diabetic Mice Exposed to Healthy Peripheral Circulation(Nature Publishing Group, 2009) Pietramaggiori, Giorgio; Scherer, Sandra S; Alperovich, Michael; Chen, Bing; Orgill, Dennis; Wagers, AmyImpaired repair of skin defects is a major complication of diabetes; yet, the pathophysiology of diabetic (db) wound healing remains largely opaque. Here, we investigate the role of humoral factors in modulating db wound repair by generating chimeric animals through parabiotic joining of wild-type (wt) and diabetic (db/db) mice. This strategy allows wounds on healing-deficient db/db mice to be exposed to factors derived from the wt circulation at physiologically appropriate concentrations. When compared with db controls, chimeric db/db animals showed significantly improved healing of full-thickness, cutaneous wounds, with enhanced granulation tissue formation, angiogenesis, cell proliferation, and collagen deposition. Glycemic control was unaffected by parabiosis; however, the distribution of circulating leukocytes, altered in db controls, normalized in db-chimeras. Both wt and db cells were recruited from circulation into db wounds, but wt cells never exceeded 20% of total cells. Improved angiogenesis persisted in db-chimeras separated 24 hours after wounding, suggesting the existence of long-term normalizing factors. This study establishes a new model for studying db wound healing, and shows a key role for circulating factors in normalizing wound repair in diabetes.