Citation: Voellenkle, C.; Fuschi, P.; Mutoli, M.; Carrara, M.; Righini, P.; Nano, G.; Gaetano, C.; Martelli, F. CircANKRD12 Is Induced in Endothelial Cell Response to Oxidative Stress. Cells 2022, 11, 3546. https://doi.org/10.3390/cells11223546 Academic Editor: Andreas Fischer Received: 13 October 2022 Accepted: 7 November 2022 Published: 9 November 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). cells Article CircANKRD12 Is Induced in Endothelial Cell Response to Oxidative Stress Christine Voellenkle 1 , Paola Fuschi 1 , Martina Mutoli 1 , Matteo Carrara 1 , Paolo Righini 2 , Giovanni Nano 2,3 , Carlo Gaetano 4 and Fabio Martelli 1, * 1 Molecular Cardiology Laboratory, IRCCS Policlinico San Donato, San Donato Milanese, 20097 Milan, Italy 2 Operative Unit of Vascular & Endovascular Surgery, IRCCS Policlinico San Donato, San Donato Milanese, 20097 Milan, Italy 3 Department of Biomedical Sciences for Health, University of Milan, 20133 Milan, Italy 4 Laboratorio di Epigenetica, Istituti Clinici Scientifici Maugeri IRCCS, 27100 Pavia, Italy * Correspondence: fabio.martelli@grupposandonato.it; Tel.: +39-0226437762 Abstract: Redox imbalance of the endothelial cells (ECs) plays a causative role in a variety of car- diovascular diseases. In order to better understand the molecular mechanisms of the endothelial response to oxidative stress, the involvement of circular RNAs (circRNAs) was investigated. Cir- cRNAs are RNA species generated by a “back-splicing” event, which is the covalent linking of the 3 ′ - and 5 ′ -ends of exons. Bioinformatics analysis of the transcriptomic landscape of human ECs exposed to H 2 O 2 allowed us to identify a subset of highly expressed circRNAs compared to their linear RNA counterparts, suggesting a potential biological relevance. Specifically, circular Ankyrin Repeat Domain 12 (circANKRD12), derived from the junction of exon 2 and exon 8 of the ANKRD12 gene (hsa_circ_0000826), was significantly induced in H 2 O 2 -treated ECs. Conversely, the linear RNA isoform of ANKRD12 was not modulated. An increased circular-to-linear ratio of ANKRD12 was also observed in cultured ECs exposed to hypoxia and in skeletal muscle biopsies of patients affected by critical limb ischemia (CLI), two conditions associated with redox imbalance and oxidative stress. The functional relevance of circANKRD12 was shown by the inhibition of EC formation of capillary-like structures upon silencing of the circular but not of the linear isoform of ANKRD12. Bioinformatics analysis of the circANKRD12–miRNA–mRNA regulatory network in H 2 O 2 -treated ECs identified the enrichment of the p53 and Foxo signaling pathways, both crucial in the cellular response to redox imbalance. In keeping with the antiproliferative action of the p53 pathway, circANKRD12 silencing inhibited EC proliferation. In conclusion, this study indicates circANKRD12 as an important player in ECs exposed to oxidative stress. Keywords: endothelium; circular RNAs; oxidative stress; circRNA–miRNA–mRNA regulatory network; p53 signaling pathway; Foxo signaling pathway 1. Introduction Reactive oxygen species (ROS) are essential mediators of both physiological and physio-pathologic signaling in the endothelium. The effects of ROS on cellular function depend on various parameters, including the localization, concentration and exposure duration of the stimulus. Complex cellular stress response mechanisms have evolved to attenuate the detrimental effects of oxidative stress. Indeed, an imbalance in redox homeostasis can lead to oxidative stress, which has a causative role in many vascular diseases [1,2]. EC oxidative stress is triggered by oxidants produced from activated immune cells or as a result of increased production of ROS from intracellular enzymes such as uncou- pled nitric oxide synthase 3 (eNOS) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, as well as from mitochondrial respiration, in the absence of neutralizing antioxidant mechanisms. Cells 2022, 11, 3546. https://doi.org/10.3390/cells11223546 https://www.mdpi.com/journal/cells