Macrophage Rac1-IL-1b Signaling in the Context of Atherosclerotic Calcification and COVID-19 Infection

Lee, Cadence F.

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Overview

Year:: 2021
Contributor:: Lee, Cadence F (creator); Morrison, Alan (Advisor); Harrington, Elizabeth (Reader); Choudhary, Gaurav (Reader); Brown University. Department of Molecular Pharmacology, Physiology and Biotechnology (sponsor)
Genre:: theses
Subject:: Atherosclerosis; COVID-19
Extent:: , None p.

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Abstract:: Abstract: Macrophage Rac1-IL-1b Signaling in the Context of Atherosclerotic Calcification and COVID-19 Infection, by Cadence Lee, ScM, Brown University, October, 2021 Objective: Previous models of progressive atherosclerotic calcification developed by the Morrison laboratory revealed that those models depend on macrophage Rac1 activity stimulating increased IL-1β expression. We sought to define the mechanisms governing Rac1-induced IL-1β expression and whether this pathway is involved in the natural progression of atherosclerosis in an experimental model of hyperlipidemia (ApoE-/-). Moreover, we seek to define the impact of this inflammatory signaling pathway in the context of coronary artery disease (CAD) and COVID-19 infection. Methods: We used novel mouse strains with tamoxifen-inducible, myeloid-specific knockouts of Rac1 and IL-1β. Animals were bred on an apolipoprotein E deficient, (ApoE−/−) atherogenic background and fed a cholesterol supplemented, high fat diet for 18-20 weeks. Aortas were analyzed ex vivo for calcification by near-infrared conjugated bisphosphonate or Alizarin red histology. Primary bone marrow-derived macrophages (BMDMs) were treated in culture with LPS+Cholesterol to mimic physiological inflammasome stimulation. Subsequent analysis involved cytokine ELISA, NF-κB and ROS assays, chromatin immunoprecipitation, and plasmid transfection. Due to limitations inherent in current animal models of SARS-CoV-2 infection, we generated a “humanized” ACE2 (hACE2) mouse strain, inserting human ACE2 mRNA sequence into the native mouse ACE2 locus under regulation of its native promoter using CRISPR-Cas9 technology. Results: Results confirm atherosclerotic calcification to be dependent on Rac1 as an upstream effector of IL-1β-mediated atherosclerotic calcification. We further demonstrate that Rac1 acts through transcription factor NF-κB to mediate IL-1β expression. We have successfully cloned the hACE2 gene into appropriate constructs for CRISPR-Cas9 mediated gene editing and have established F0 founders and F1 progeny, demonstrating successful breeding and that the hACE2 allele is passed on in the germ line. Conclusions: Rac1 acts through NF-κB to increase IL-1β-dependent atherosclerotic calcification. A novel humanized ACE2 mouse strain is being developed and will help to define the mechanism of CAD-mediated worsening outcomes in COVID-19 infection.
Notes:: Thesis (Sc. M.)--Brown University, 2021

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Citation

Lee, Cadence F., "Macrophage Rac1-IL-1b Signaling in the Context of Atherosclerotic Calcification and COVID-19 Infection" (2021). Molecular Pharmacology, Physiology, and Biotechnology Theses and Dissertations. Brown Digital Repository. Brown University Library. https://repository.library.brown.edu/studio/item/bdr:xwktmh3b/

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Molecular Pharmacology, Physiology, and Biotechnology Theses and Dissertations

Theses and Dissertations for the Molecular Pharmacology, Physiology, and Biotechnology department.
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