RNA editing controls polyvascular disease by enhancing metabolic inflammation

G. Rackov (Mannheim)¹, M. Amponsah-Offeh (Mannheim)², G. Georgiopoulos (Athens)³, G. Ciliberti (Mannheim)⁴, M. Sachse (Mannheim)², G. Mavraganis (Athens)³, G. Zervas (Athens)⁵, M. Polycarpou-Schwarz (Mannheim)², K. Sopova (Mannheim)⁶, M. Sigl (Mannheim)⁷, E. Lutgens (Rochester, MN)⁸, S. Dimmeler (Frankfurt am Main)⁹, K. Stamatelopoulos (Athens)³, S. Tual-Chalot (Newcastle Upon Tyne)¹⁰, K. Stellos (Mannheim)^11
1European Center for Angioscience Department of Cardiovascular Research Mannheim, Deutschland; ²Medizinische Fakultät Mannheim der Universität Heidelberg Abteilung für Herz- Kreislaufforschung Mannheim, Deutschland; ³University of Athens Department of Clinical Therapeutics Athens, Griechenland; ⁴Universitätsklinik Mannheim Abteilung für Herz- und Kreislaufforschung Mannheim, Deutschland; ⁵National and Kapodistrian University of Athens Medical School Department of Clinical Therapeutics Athens, Deutschland; ⁶Universitätsklinikum Mannheim GmbH I. Medizinische Klinik Mannheim, Deutschland; ⁷University Medical Centre Mannheim Department of Medicine VI Mannheim, Deutschland; ⁸Mayo Clinic, Rochester, MN Department of Cardiovascular Medicine, Experimental Cardiovascular Immunology Laboratory, Rochester, MN, USA; ⁹Goethe Universität Frankfurt am Main Zentrum für Molekulare Medizin, Institut für Kardiovaskuläre Regeneration Frankfurt am Main, Deutschland; ¹⁰Newcastle University Biosciences Institute Newcastle Upon Tyne, Großbritannien; ¹¹Universitätsmedizin Mannheim der Universität Heidelberg Institut für Herz-Kreislaufforschung Mannheim, Deutschland

Introduction Post-transcriptional regulation of gene expression by RNA-based mechanisms, like adenosine-to-inosine RNA editing, can overwrite transcriptional programs and determine cell signaling in disease. Whether the enzyme ADARB1 that catalyzes the deamination of adenosine to inosine in RNA molecules, is involved in atherosclerosis remains elusive.
Methods Expression levels of ADARB1 were measured in peripheral blood mononuclear cells derived from 606 individuals at risk for atherosclerotic vascular disease. Structural vascular measurements were used, including intima-media thickness (IMT), carotid wall maximum thickness (maxWT), and the number of atheromatous plaques by carotid and femoral artery ultrasonography as surrogate markers of subclinical arterial disease. Study participants were followed for major adverse cardiovascular events (MACE). Experimental atherosclerosis was assessed in double ADARB1 and ApoE knockout mice fed with a Western diet for 20 weeks. Blood levels of lipids and cytokines were measured in plasma by Luminex assays. Immunophenotyping of aortic plaques was performed using imaging by mass cytometry.
Results In humans, increased ADARB1 mRNA expression was independently associated with increased hs-CRP>2mg/dl, a marker of metabolic inflammation driven by elevated IL-6 production, and with the presence of coronary artery disease. Increased ADARB1 mRNA expression was independently associated with polyvascular disease defined by the number of affected vascular beds: a) presence of a plaque in carotid arteries, b) >50% stenosis in a coronary artery, c) increased pulse wave velocity>10m/sec in aorta, and d) presence of a plaque in femoral arteries at baseline. Increased ADARB1 expression was associated with accelerated burden of atheromatosis in the carotid/femoral arteries or the carotid arteries after a median follow-up of 33 months (OR=3.74, P<0.05). Patients at the highest ADARB1 tertile had a higher incidence of major adverse cardiovascular events (MACE), compared to their counterparts in the lower tertiles. Mechanistically, ADARB1 knockout (KO) mice exhibited attenuated atherosclerotic lesion formation in the aorta and brachiocephalic artery, and presented reduced features of plaque vulnerability, including smaller necrotic cores relative to control mice. These effects were independent of plasma lipid levels since the cholesterol and triglyceride profiles of these animals were similar. Absence of ADARB1 in atherosclerotic mice reduced the levels of several circulating pro-inflammatory mediators, especially interleukin-6. A Western-type diet led to a 2.3-fold increase in IL-6 levels in wild-type animals, an effect that was abolished in the absence of ADARB1. Similarly, the increase of circulating monocytes and neutrophil counts observed after a Western-type diet was markedly reduced after ADARB1 depletion, suggesting a possible effect of the ADARB1/IL-6 axis on hematopoiesis in atherosclerosis.
Conclusion Our findings indicate that ADARB1 controls atherosclerosis by driving metabolic inflammation through feed-forward inflammatory loops, which include IL-6–driven inflammatory hematopoiesis. Post-transcriptional regulation of IL-6 by ADAR2 emerges as a promising therapeutic target to mitigate the high residual inflammatory risk in atherosclerosis.