Crispr/dcas9 transcriptional activation of endogenous apolipoprotein ai and paraoxonase 1 in enterocytes alleviates endothelial cell dysfunction

Abstract

Atherosclerosis is the main cause of cardiovascular diseases with high prevalence world-wide. A promising therapeutic strategy to reverse atherosclerotic process is to improve the athero-protective potential of high-density lipoproteins (HDL). Since the small intestine is a source of HDL, we aimed to activate transcription of the endogenous HDL major proteins, apolipoprotein AI (ApoAI) and paraoxonase 1 (PON1), in enterocytes, and to evaluate their potential to correct the pro-inflammatory status of endothelial cells (EC). Caco-2 enterocytes were transfected with CRISPR activation plasmids targeting ApoAI or PON1, and their gene and protein expression were meas-ured in cells and conditioned medium (CM). ATP binding cassette A1 and G8 transporters (ABCA1, ABCG8), scavenger receptor BI (SR-BI), and transcription regulators peroxisome proliferator-acti-vated receptor γ (PPARγ), liver X receptors (LXRs), and sirtuin-1 (SIRT1) were assessed. Anti-in-flammatory effects of CM from transfected enterocytes were estimated through its ability to inhibit tumor necrosis factor α (TNFα) activation of EC. Transcriptional activation of ApoAI or PON1 in enterocytes induces: (i) increase of their gene and protein expression, and secretion in CM; (ii) stim-ulation of ABCA1/G8 and SR-BI; (iii) upregulation of PPARγ, LXRs, and SIRT1. CM from trans-fected enterocytes attenuated the TNFα-induced inflammatory and oxidative stress in EC, by de-creasing TNF receptor 1, monocyte chemoattractant protein-1, and p22phox. In conclusion, transcriptional activation of endogenous ApoAI or PON1 in enterocytes by CRISPR/dCas9 system is a realistic approach to stimulate biogenesis and function of major HDL proteins which can regulate cholesterol efflux transporters and reduce the inflammatory stress in activated EC.