Correspondence Article Type Response to Letter Regarding Article “Role of Extracellular RNA in Atherosclerotic Plaque Formation in Mice” We acknowledge the interest and comments of Dr Chen et al regarding our findings that self-extracellular RNA (eRNA) significantly contributes to atherogenesis (as demonstrated in 2 established animal models) by inducing a prominent inflammatory response in situ and in bone marrow–derived macrophages (BMDM), as well.1 In particular, Chen et al question whether eRNA-dependent effects may have been mediated by Toll-like receptor (TLR)–related signaling, because they recently reported that BMDM responses toward the RNA analogue poly(IC) were significantly dampened in TLR3deficient cells.2 During the past decade, our laboratory has characterized a number of new functions of eRNA in inflammation and cardiovascular diseases. In the indicated study, we aimed to characterize a causal role of natural eRNA, which may also serve as a cell-injury marker, in the onset and progression of atherosclerosis. It was not our purpose to mimic a viral infection by using poly(IC) as an artificial analogue of double-stranded viral RNA. To this end, self-eRNA (composed mainly of ribosomal RNA) was used for all in vitro and cellstimulating experiments, whereas Chen et al applied high doses of poly(IC) (20–25 μg/mL) to stimulate BMDM in their study,2 which, based on our experience, can cause cytotoxicity at such high doses. Following our rationale, we have not specifically tested whether eRNA exposure of BMDM may lead to an increase in type I interferon expression, as reported for poly(IC). Our robust eRNA-related cytokine responses in BMDM (including the release of tumor necrosis factor-α and activation of nuclear factor κB signaling) could not be duplicated by the TLR3-agonist (stimulation with 10 μg/mL of poly(IC)) or single-stranded RNA as agonists for TLR7 or TLR8. Rather, a most recent study from our laboratory demonstrates that a particular lectin expressed on BMDM appears to be responsible for the interaction with self-eRNA.3 Nevertheless, we concur that different kinds of eRNA need to be tested in future studies as to the respective cellular recognition/receptors involved, the underlying intracellular signaling pathway(s) engaged, and the functional outcome of the cellular induction mechanisms. It thus may very well be that minor fractions of eRNA such as micro-RNA or longnoncoding RNA become recognized by TLRs, whereas the majority of self-eRNA can foster strong cellular responses independent of typical pattern-recognition receptors. Notably, although Chen et al2 observed the release of RNA from necrotic macrophages and cardiomyocytes in vitro and after transient myocardial ischemia in vivo, deficiency in TLR3 had no impact on cardiac cytokine production in vivo, including interferon responses. This observation is in favor of our findings that self-eRNA contributes to inflammatory responses, mostly independent of TLR3. eRNA can trigger proteolytic shedding of soluble tumor necrosis factor-α from its transmembrane proform (eg, on macrophages) such that bioactive tumor necrosis factor-α may in turn amplify cell stimulation in an autocrine fashion.4 Likewise, we reported that eRNA is required to induce vascular permeability via the mobilization/stabilization and direct binding to vascular endothelial growth factor.5 These findings epitomize that self-eRNA may furthermore promote inflammatory responses in different cell types also in an indirect fashion.

Disclosures None.

Sakine Simsekyilmaz, PhD Institute for Molecular Cardiovascular Research RWTH University Hospital Aachen, Germany Hector A. Cabrera-Fuentes, PhD Department of Biochemistry Medical School Justus-Liebig-University Giessen, Germany Department of Microbiology Kazan Federal University Kazan, Russian Federation Svenja Meiler, PhD Center for Cardiovascular Research John A. Burns School of Medicine University of Hawaii Honolulu, Hawaii Sawa Kostin, PhD, MD Core Lab for Molecular and Structural Biology Max-Planck-Institute for Heart and Lung Research Bad Nauheim, Germany Yvonne Baumer, PhD Center for Cardiovascular Research John A. Burns School of Medicine University of Hawaii Honolulu, Hawaii Elisa A. Liehn, MD, PhD Institute for Molecular Cardiovascular Research RWTH University Hospital Aachen, Germany Christian Weber, MD Institute for Cardiovascular Prevention Ludwig-Maximilians-University Munich, Germany William A. Boisvert, PhD Center for Cardiovascular Research John A. Burns School of Medicine University of Hawaii Honolulu, Hawaii Klaus T. Preissner, PhD Department of Biochemistry Medical School Justus-Liebig-University Giessen, Germany Alma Zernecke, MD Institute of Clinical Biochemistry and Pathobiochemistry University Hospital Würzburg, Germany

References 1. Simsekyilmaz S, Cabrera-Fuentes HA, Meiler S, Kostin S, Baumer Y, Liehn EA, Weber C, Boisvert WA, Preissner KT, Zernecke A. Role of extracellular RNA in atherosclerotic plaque formation in mice. Circulation. 2014;129:598–606.

(Circulation. 2014;130:e144-e145.) © 2014 American Heart Association, Inc. Circulation is available at http://circ.ahajournals.org

DOI: 10.1161/CIRCULATIONAHA.114.012346

Downloaded from http://circ.ahajournals.org/ at New York University/ Medical Center--New York on January 11, 2015 e144

Correspondence  e145 2. Chen C, Feng Y, Zou L, Wang L, Chen HH, Cai JY, Xu JM, Sosnovik DE, Chao W. Role of extracellular RNA and TLR3-Trif signaling in myocardial ischemia-reperfusion injury. J Am Heart Assoc. 2014; 3:e000683. 3. Cabrera-Fuentes HA, Galuska S, Meiler S, Baumer Y, Mccurdy S, Fischer S, Preissner K, Boisvert W. Regulation of macrophage polarization by extracellular RNA: the role of sialoadhesin-1. Cardiovasc Res. 2014;103(suppl 1):S135.

4. Fischer S, Gesierich S, Griemert B, Schänzer A, Acker T, Augustin HG, Olsson AK, Preissner KT. Extracellular RNA liberates tumor necrosis factor-α to promote tumor cell trafficking and progression. Cancer Res. 2013;73:5080–5089. 5. Fischer S, Gerriets T, Wessels C, Walberer M, Kostin S, Stolz E, Zheleva K, Hocke A, Hippenstiel S, Preissner KT. Extracellular RNA mediates endothelial-cell permeability via vascular endothelial growth factor. Blood. 2007;110:2457–2465.

Downloaded from http://circ.ahajournals.org/ at New York University/ Medical Center--New York on January 11, 2015

Response to Letter Regarding Article ''Role of Extracellular RNA in Atherosclerotic Plaque Formation in Mice'' Sakine Simsekyilmaz, Hector A. Cabrera-Fuentes, Svenja Meiler, Sawa Kostin, Yvonne Baumer, Elisa A. Liehn, Christian Weber, William A. Boisvert, Klaus T. Preissner and Alma Zernecke Circulation. 2014;130:e144-e145 doi: 10.1161/CIRCULATIONAHA.114.012346 Circulation is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 2014 American Heart Association, Inc. All rights reserved. Print ISSN: 0009-7322. Online ISSN: 1524-4539

The online version of this article, along with updated information and services, is located on the World Wide Web at: http://circ.ahajournals.org/content/130/16/e144

Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in Circulation can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office. Once the online version of the published article for which permission is being requested is located, click Request Permissions in the middle column of the Web page under Services. Further information about this process is available in the Permissions and Rights Question and Answer document. Reprints: Information about reprints can be found online at: http://www.lww.com/reprints Subscriptions: Information about subscribing to Circulation is online at: http://circ.ahajournals.org//subscriptions/

Downloaded from http://circ.ahajournals.org/ at New York University/ Medical Center--New York on January 11, 2015