Disrupting the vicious cycle created by NOX activation in sickle erythrocytes exposed to hypoxia/reoxygenation prevents adhesion and vasoocclusion.

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2019-07

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Abstract

In sickle cell disease (SCD), recurrent painful vasoocclusive crisis are likely caused by repeated episodes of hypoxia and reoxygenation. The sickle erythrocyte (SSRBC) adhesion plays an active role in vasoocclusion. However, the effect of prolonged reoxygenation after hypoxic stress on the molecular mechanisms in SSRBCs involved in onset of episodic vasoocclusion remain unclear. Exposure of human SSRBCs to hypoxia followed by 2 h reoxygenation, increased reactive oxygen species (ROS) production. Using specific pharmacological inhibitors, we show that excess ROS production in both reticulocytes and mature SSRBCs is regulated by NADPH oxidases (NOXs), the mitogen-activated protein kinase (ERK1/2), and G-protein coupled-receptor kinase 2 (GRK2). Consequently, SSRBC ROS create an intracellular positive feedback loop with ERK1/2 and GRK2 to mediate SSRBC adhesion to endothelium in vitro, and vasoocclusion in a mouse model of vasoocclusion in vivo. Importantly, reducing ROS levels in SSRBCs with redox-active manganese (Mn) porphyrins, commonly known as mimics of superoxide dismutase (SOD), disrupted the cycle created by ROS by affecting NOX and GRK2 activities and ERK1/2 phosphorylation, thus abrogating RBC-endothelial interactions. Inhibition adhesion assays show that LW (ICAM-4, CD242) blood group glycoprotein and CD44 are the RBC adhesion molecules mediating endothelial binding. Conversely, hypoxia/reoxygenation of normal RBCs failed to activate this feedback loop, and adhesion. These findings provide novel insights into the pathophysiological significance of the deleterious cycle created by NOX-dependent ROS, GRK2 and ERK1/2 within SSRBCs activated by hypoxia/reoxygenation, and involved in SSRBC adhesion and vasoocclusion. Thus, this loop in SSRBCs, which can be disrupted by Mn porphyrins, likely drives the profound SCD vasculopathy, and may point to new therapeutic targets to prevent chronic vasoocclusive events.

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10.1016/j.redox.2019.101097

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MacKinney, Anson, Emily Woska, Ivan Spasojevic, Ines Batinic-Haberle and Rahima Zennadi (2019). Disrupting the vicious cycle created by NOX activation in sickle erythrocytes exposed to hypoxia/reoxygenation prevents adhesion and vasoocclusion. Redox biology, 25. p. 101097. 10.1016/j.redox.2019.101097 Retrieved from https://hdl.handle.net/10161/22408.

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Scholars@Duke

Spasojevic

Ivan Spasojevic

Associate Professor in Medicine
Batinic-Haberle

Ines Batinic-Haberle

Professor Emeritus of Radiation Oncology

            A major interest of mine has been in the design and synthesis of Mn porphyrin(MnP)-based powerful catalytic antioxidants which helped establish structure-activity relationship (SAR). It relates the redox property of metalloporphyrins to their ability to remove superoxide. SAR has facilitated the design of redox-active therapeutics and served as a tool for mechanistic considerations. Importantly SAR parallels the magnitude of the therapeutic potential of SOD mimics and is valid for all classes of redox-active compounds. Two lead Mn porphyrins are already in five Phase II clinical trials (reviewed in Batinic-Haberle et al, Oxid Med Cell Longevity 2021). Recent research suggests immense potential of MnPs in cardiac diseases. MnTE-2-PyP (AEOL10113, BMX-010) prevents and treats cardiac arrhythmia, while MnTnBuOE-2-PyP (BMX-001) fully suppressed the development of aortic sclerosis in mice. The latter result is relevant to the cancer patients undergoing chemotherapy. In addition to breast cancer, in collaboration with Angeles Alvarez Secord, MD, MHSc, we have recently shown the anticancer effects of Mn porphyrin/ascorbate in cellular and mouse models of ovarian cancer.

            In parallel with synthetic efforts, I have also been interested in the mechanistic aspects of differential actions of Mn porphyrins in normal vs tumor tissue. In-depth studies of chemistry and biology of the reactions of MnPs with redox-active agents relevant to cancer therapy – ascorbate, chemotherapy and radiation – set ground for understanding the role of thermodynamics and kinetics in the mechanism of action of Mn porphyrins. Mechanistic studies have been revealed in Batinic-Haberle et al, Antioxidant Redox Signal 2018, Batinic-Haberle and Tome, Redox Biology 2019 and Batinic-Haberle et al Oxidative Medicine and Cellular Longevity 2021. My research has resulted in over 230 publications, 18 268 citations and an h-index of 64. For my achievements, I have been awarded the 2021 Discovery Award from the Society for Redox Biology and Medicine, SfRBM.

Additional Training

  • Postdoctoral fellowship with Professor Alvin Crumbliss in the field of Bioinorganic Chemistry, Department of Chemistry, Duke University
  • Postdoctoral fellowship with Professor Irwin Fridovich in the field of Redox Biology, Department of Biochemistry, Duke University School of Medicine

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