Citation: Huang, K.; Pitman, M.; Oladosu, O.; Echesabal-Chen, J.; Vojtech, L.; Esobi, I.; Larsen, J.; Jo, H.; Stamatikos, A. The Impact of MiR-33a-5p Inhibition in Pro-Inflammatory Endothelial Cells. Diseases 2023, 11, 88. https:// doi.org/10.3390/diseases11030088 Academic Editors: Diana Díaz-García and Santiago Gómez-Ruiz Received: 13 May 2023 Revised: 18 June 2023 Accepted: 21 June 2023 Published: 24 June 2023 Copyright: © 2023 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/). diseases Article The Impact of MiR-33a-5p Inhibition in Pro-Inflammatory Endothelial Cells Kun Huang 1 , Mark Pitman 2 , Olanrewaju Oladosu 1 , Jing Echesabal-Chen 1 , Lucia Vojtech 3 , Ikechukwu Esobi 1 , Jessica Larsen 2,4 , Hanjoong Jo 5 and Alexis Stamatikos 1, * 1 Department of Food, Nutrition, and Packaging Sciences, Clemson University, Clemson, SC 29634, USA; kunh@g.clemson.edu (K.H.); oolados@g.clemson.edu (O.O.); jchen11@clemson.edu (J.E.-C.); iesobi@g.clemson.edu (I.E.) 2 Department of Chemical and Biomolecular Engineering, Clemson University, Clemson, SC 29634, USA; pitman@g.clemson.edu (M.P.); larsenj@clemson.edu (J.L.) 3 Department of Obstetrics & Gynecology, University of Washington, Seattle, WA 98109, USA; luciav@uw.edu 4 Department of Bioengineering, Clemson University, Clemson, SC 29634, USA 5 Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30322, USA; hjo@emory.edu * Correspondence: adstama@clemson.edu Abstract: Evidence suggests cholesterol accumulation in pro-inflammatory endothelial cells (EC) contributes to triggering atherogenesis and driving atherosclerosis progression. Therefore, inhibit- ing miR-33a-5p within inflamed endothelium may prevent and treat atherosclerosis by enhancing apoAI-mediated cholesterol efflux by upregulating ABCA1. However, it is not entirely elucidated whether inhibition of miR-33a-5p in pro-inflammatory EC is capable of increasing ABCA1-dependent cholesterol efflux. In our study, we initially transfected LPS-challenged, immortalized mouse aortic EC (iMAEC) with either pAntimiR33a5p plasmid DNA or the control plasmid, pScr. We detected significant increases in both ABCA1 protein expression and apoAI-mediated cholesterol efflux in iMAEC transfected with pAntimiR33a5p when compared to iMAEC transfected with pScr. We subse- quently used polymersomes targeting inflamed endothelium to deliver either pAntimiR33a5p or pScr to cultured iMAEC and showed that the polymersomes were selective in targeting pro-inflammatory iMAEC. Moreover, when we exposed LPS-challenged iMAEC to these polymersomes, we observed a significant decrease in miR-33a-5p expression in iMAEC incubated with polymersomes containing pAntimR33a5p versus control iMAEC. We also detected non-significant increases in both ABCA1 pro- tein and apoAI-mediated cholesterol in iMAEC exposed to polymersomes containing pAntimR33a5p when compared to control iMAEC. Based on our results, inhibiting miR-33a-5p in pro-inflammatory EC exhibits atheroprotective effects, and so precisely delivering anti-miR-33a-5p to these cells is a promising anti-atherogenic strategy. Keywords: endothelial activation; endothelial dysfunction; HDL; microRNA; nanoparticle; nanother- apy; reverse cholesterol transport; vascular inflammation; VCAM-1 1. Introduction Atherosclerotic cardiovascular disease is a condition that kills more people worldwide than any other disease [1]. While the pathophysiology of atherosclerotic cardiovascular disease is complex and poorly understood [2], endothelial cell (EC) inflammation is rec- ognized to contribute to both atherogenesis and atherosclerosis progression [3]. Indeed, vascular inflammation is an intriguing component of atherosclerosis, as inflammation in the EC facilitates monocyte/macrophage intimal entry, which allows macrophages to engulf intimal cholesterol [4]. This in turn may result in the accumulation of lipid-laden macrophages within the intima, leading to atherosclerosis formation [5]. Endothelial dysfunction and activation are strongly linked with EC inflammation [6,7]. Interestingly, an acknowledged precursor to endothelial dysfunction/activation is EC Diseases 2023, 11, 88. https://doi.org/10.3390/diseases11030088 https://www.mdpi.com/journal/diseases