Discipline: Biological Sciences
Subcategory: Physiology and Health
KiAundra L. Kilpatrick - Florida Agricultural and Mechanical University
Co-Author(s): Ryan A. Frieler, Ph.D., University of Michigan, Ann Arbor, MI; Thomas M. Vigil, University of Michigan, Ann Arbor, MI; Richard Mortensen, M.D., Ph.D., University of Michigan, Ann Arbor, MI
Stroke is a leading cause of death and permanent disability in the United States, but there remain very few treatment options highlighting a need for new therapeutic strategies. Succinate accumulation during ischemia followed by rapid metabolism during reperfusion has recently been shown to be a major cause of reactive oxygen species production and subsequent tissue injury during stroke. The citric acid cycle and succinate metabolism have also been shown to have important roles in macrophage activation and inflammation, and succinate metabolism can be regulated by targeting the enzyme succinate dehydrogenase. Itaconate is produced from TCA cycle intermediates by the enzyme aconitate decarboxylase (ACOD1) and it decreases oxidative phosphorylation through succinate dehydrogenase inhibition. More importantly, administration of exogenous itaconate has been shown to be protective in a cardiac model of ischemia-reperfusion injury. To test the role of itaconate during stroke, we used ACOD1 deficient mice to disrupt endogenous itaconate synthesis and also tested the effect of administration of dimethyl itaconate in a cerebral ischemia reperfusion model. We further investigated the effects of dimethyl itaconate administration during hypoxia induced inflammation in macrophage cell lines. ACOD1 deficient mice (ACOD1-/-) and wild type controls on a C57BL/6N background were subjected to transient 90-minute middle cerebral artery occlusion and infarct size was assessed by MRI after 24 hours of reperfusion. Dimethyl itaconate (DMI, 210 mg/kg) was administered before the 90-minute period of ischemia and prior to reperfusion. Murine macrophage cell lines (RAW 264.7 and J774) were activated with lipopolysaccharide (LPS) and tested under normoxic and hypoxic conditions. We found that ACOD1-/- mice had significantly increased infarct size (ACOD1-/-: 77.8 ±6.5 mm3, Control: 56.0 ±6.6mm3, P < 0.05) in comparison to control mice. In our preliminary results, administration of DMI did not significantly decrease infarct size (P = 0.08) but was suggestive of a protective effect. DMI treatment (250 µM) in macrophage cell lines significantly decreased LPS-induced IL-1gene expression. Furthermore, under hypoxic and LPS-activating conditions, administration of DMI increased the expression of the angiogenic factor VEGF-A. These data show that disruption of endogenous itaconate synthesis through ACOD1 knockout is harmful during stroke indicating a potentially beneficial role for itaconate with promise for therapeutic intervention. These data also indicate a potential role for macrophages in mediating this effect.
Funder Acknowledgement(s): NIH Grant # R01-HL112610
Faculty Advisor: Richard Mortensen, M.D., Ph.D., email@example.com
Role: During the course of this research project, I cultured and harvested the murine macrophage cell lines (J774 and RAW 264.7) that were used in the in-vitro hypoxia-reoxygenation experiments. I administered all treatments for the in-vitro experiment (Lipopolysaccharide (LPS) to activate macrophage cell lines), Dimethyl Itaconate (DMI) to test the effects of exogenous itaconate in activated macrophages under normoxic and hypoxic conditions, and hypoxia chamber treatment (to induce hypoxia and create an in-vitro model of ischemia). Following the treatments, I conducted RNA isolation for each cell line and quantified levels of gene expression for select genes (IL-1b (beta) and VEGFa) using reverse transcription and quantitative polymerase chain reaction (qPCR).