Browsing by Subject "Physioxia"

Now showing 1 - 5 of 5
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    Characterization of Normal and Preleukemic Hematopoietic Stem Cell Responses to Physiologic and Extra-Physiologic Oxygen Tension
    (2022-08) Aljoufi, Arafat; Kaplan, Mark H.; Zhang, Chi; Srour, Edward F.; Kapur, Reuben
    Hematopoietic stem and progenitor cells (HSCs/HPCs) transplantation is a curative treatment for a variety of hematologic and non-hematologic diseases. Successful HSC transplantation requires infusing patients with a sufficient number of long-term engrafting HSCs. As a result, research efforts have focused on optimizing the collection process. Previous work established that harvesting mouse bone marrow HSCs under low oxygen tension similar to that reported for the bone marrow niche in situ (physioxia), results in enhanced HSC recovery and function. However, collecting bone marrow cells under physioxia is not a clinically viable approach. Here, I demonstrated that the collection and processing of peripheral blood mobilized with G-CSF alone or G-CSF and Plerixafor under physioxia resulted in a greater number of phenotypically defined long-term engrafting HSCs. Using high-resolution single cell sequencing to explore the molecular programs governing HSCs under physioxia, I identified increased expression of genes involved in HSC self-renewal and maintenance. In contrast, HSCs under ambient air upregulated genes implicated in HSC differentiation, apoptosis, and inflammatory pathways. Furthermore, wild-type HSCs under physioxia revealed a significant reduction in gene expression and activity of the epigenetic modifier Tet2. Consequently, I evaluated the phenotyping, engraftment potential and gene expression of preleukemic Tet2-/- bone marrow cells under physioxia and ambient air. Unlike wild-type HSCs, Tet2-/- HSCs/HPCs were unresponsive to changes in oxygen tension. Notably, we observed similar phenotypes, functions, and self-renewal and quiescence gene expression in wild-type HSCs under physioxia and Tet2- /- HSCs under physioxia or ambient air. These findings imply that the preserved stemness and enhanced engraftment of HSCs under physioxia may in part be a result of Tet2 downregulation. Understanding the mechanisms regulating wild-type and preleukemic HSCs under physioxia will have therapeutic implications for optimizing HSC transplantation and mitigating the growth advantage of preleukemic stem cells.
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    Hypoxia-Inducible Factor 1α Stabilization Restores Epigenetic Control of Nitric Oxide Synthase 1 Expression and Reverses Gastroparesis in Female Diabetic Mice
    (Elsevier, 2023) Gao, Fei; Hayashi, Yujiro; Saravanaperumal, Siva Arumugam; Gajdos, Gabriella B.; Syed, Sabriya A.; Bhagwate, Aditya V.; Ye, Zhenqing; Zhong, Jian; Zhang, Yuebo; Choi, Egan L.; Kvasha, Sergiy M.; Kaur, Jagneet; Paradise, Brooke D.; Cheng, Liang; Simone, Brandon W.; Wright, Alec M.; Kellogg, Todd A.; Kendrick, Michael L.; McKenzie, Travis J.; Sun, Zhifu; Yan, Huihuang; Yu, Chuanhe; Bharucha, Adil E.; Linden, David R.; Lee, Jeong-Heon; Ordog, Tamas; Medicine, School of Medicine
    Background & aims: Although depletion of neuronal nitric oxide synthase (NOS1)-expressing neurons contributes to gastroparesis, stimulating nitrergic signaling is not an effective therapy. We investigated whether hypoxia-inducible factor 1α (HIF1A), which is activated by high O2 consumption in central neurons, is a Nos1 transcription factor in enteric neurons and whether stabilizing HIF1A reverses gastroparesis. Methods: Mice with streptozotocin-induced diabetes, human and mouse tissues, NOS1+ mouse neuroblastoma cells, and isolated nitrergic neurons were studied. Gastric emptying of solids and volumes were determined by breath test and single-photon emission computed tomography, respectively. Gene expression was analyzed by RNA-sequencing, microarrays, immunoblotting, and immunofluorescence. Epigenetic assays included chromatin immunoprecipitation sequencing (13 targets), chromosome conformation capture sequencing, and reporter assays. Mechanistic studies used Cre-mediated recombination, RNA interference, and clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-mediated epigenome editing. Results: HIF1A signaling from physiological intracellular hypoxia was active in mouse and human NOS1+ myenteric neurons but reduced in diabetes. Deleting Hif1a in Nos1-expressing neurons reduced NOS1 protein by 50% to 92% and delayed gastric emptying of solids in female but not male mice. Stabilizing HIF1A with roxadustat (FG-4592), which is approved for human use, restored NOS1 and reversed gastroparesis in female diabetic mice. In nitrergic neurons, HIF1A up-regulated Nos1 transcription by binding and activating proximal and distal cis-regulatory elements, including newly discovered super-enhancers, facilitating RNA polymerase loading and pause-release, and by recruiting cohesin to loop anchors to alter chromosome topology. Conclusions: Pharmacologic HIF1A stabilization is a novel, translatable approach to restoring nitrergic signaling and treating diabetic gastroparesis. The newly recognized effects of HIF1A on chromosome topology may provide insights into physioxia- and ischemia-related organ function.
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    Modeling Preclinical Cancer Studies under Physioxia to Enhance Clinical Translation
    (American Association for Cancer Research, 2022) Adebayo, Adedeji K.; Nakshatri, Harikrishna; Surgery, School of Medicine
    Oxygen (O2) plays a key role in cellular homeostasis. O2 levels are tightly regulated in vivo such that each tissue receives an optimal amount to maintain physiologic status. Physiologic O2 levels in various organs range between 2–9% in vivo, with the highest levels of 9% in the kidneys and the lowest of 0.5% in parts of the brain. This physiologic range of O2 tensions is disrupted in pathologic conditions such as cancer, where it can reach as low as 0.5%. Regardless of the state, O2 tension in vivo is maintained at significantly lower levels than ambient O2, which is approximately 21%. Yet, routine in vitro cellular manipulations are carried out in ambient air, regardless of whether or not they are eventually transferred to hypoxic conditions for subsequent studies. Even brief exposure of hematopoietic stem cells to ambient air can cause detrimental effects through a mechanism termed extraphysiologic oxygen shock/stress (EPHOSS), leading to reduced engraftment capabilities. Here, we provide an overview of the effects of ambient air exposure on stem and non-stem cell subtypes, with a focus on recent findings that reveal the impact of EPHOSS on cancer cells.
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    Physioxia enhances T-cell development ex vivo from human hematopoietic stem and progenitor cells
    (Nova Science, 2020-11) Shin, Dong-Yeop; Huang, Xinxin; Gil, Chang-Hyun; Aljoufi, Arafat; Ropa, James; Broxmeyer, Hal E.; Microbiology and Immunology, School of Medicine
    Understanding physiologic T-cell development from hematopoietic stem (HSCs) and progenitor cells (HPCs) is essential for development of improved hematopoietic cell transplantation (HCT) and emerging T-cell therapies. Factors in the thymic niche, including Notch 1 receptor ligand, guide HSCs and HPCs through T-cell development in vitro. We report that physiologically relevant oxygen concentration (5% O2,physioxia), an important environmental thymic factor, promotes differentiation of cord blood CD34+ cells into progenitor T (proT) cells in serum-free and feeder-free culture system. This effect is enhanced by a potent reducing and antioxidant agent, ascorbic acid. Human CD34+ cell-derived proT cells in suspension cultures maturate into CD3+ T cells in an artificial thymic organoid (ATO) culture system more efficiently when maintained under physioxia, compared to ambient air. Low oxygen tension acts as a positive regulator of HSC commitment and HPC differentiation toward proT cells in the feeder-free culture system and for further maturation into T cells in the ATO. Culturing HSCs/HPCs in physioxia is an enhanced method of effective progenitor T and mature T-cell production ex vivo and may be of future use for HCT and T-cell immunotherapies.
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    Role of Tumor Oxygen Tension in Signaling and Response to Targeted Therapies
    (2024-10) Adebayo, Adedeji Kolawole; Nakshatri, Harikrishna; Quilliam, Lawrence; Capitano, Maegan; Kim, Jaeyeon
    Most tumor cells in solid tumors are exposed to oxygen levels ranging from 0.5% to 5%, but never to ambient air oxygen levels of about 21%. We developed an approach that allows collection, processing and evaluation of cancer and non-cancer cells under physioxia (3%-5% oxygen), ensuring little to no exposure to ambient air. This approach allowed for comparison of baseline and targeted therapy-induced changes in signaling pathways in cells under physioxia and ambient air and to identify potentially efficacious therapeutic combinations based on signaling pathways uniquely active under physioxia. Using tumor cells from two transgenic models of breast cancer and cells from breast tissues of clinically breast cancer-free women, we demonstrate oxygen level-dependent differences in cell preference for EGFR or PDGFRβ signaling. Physioxia caused PDGFRβ-mediated activation of AKT and ERK that reduced tumor cell sensitivity to EGFR and PIK3CA inhibition and maintained PDGFRβ+ epithelial-mesenchymal hybrid cells with potential cancer stem cell properties. Cells in ambient air displayed differential EGFR activation and were sensitive to EGFR and PIK3CA inhibition. Tumor cells grown under physioxia were sensitive to high affinity PDGFRβ inhibitor sunitinib. Furthermore, significantly higher synergistic growth inhibition and apoptosis was observed with lapatinib (a clinically used dual EGFR and ErbB2/HER2 inhibitor) and sunitinib combination only in tumor cells under physioxia both in vitro and in vivo. Our data emphasize the importance of oxygen considerations in preclinical cancer research to evaluate clinically relevant signaling pathways and identify novel drug targets or combination therapy approaches. We suggest that evaluation of candidate drugs for their efficacy under physiologic oxygen levels in preclinical models, prior to transitioning into clinical trials, would not only accelerate the development of effective drugs but also reduce failure at the clinical trial stage.