Abstract
Atherosclerosis is the main cause of cardiovascular diseases with high prevalence worldwide. 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 measured 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-activated receptor γ (PPARγ), liver X receptors (LXRs), and sirtuin-1 (SIRT1) were assessed. Anti-inflammatory 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) stimulation of ABCA1/G8 and SR-BI; (iii) upregulation of PPARγ, LXRs, and SIRT1. CM from transfected enterocytes attenuated the TNFα-induced inflammatory and oxidative stress in EC, by decreasing 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.
Highlights
Introduction published maps and institutional affilCardiovascular diseases (CVD) represent the clinical manifestation of atherosclerosis and are a major threat to public health worldwide, with high prevalence and mortality rates [1,2]
To assess whether transfection of CRISPR/dCas9 apolipoprotein AI (ApoAI) plasmids increases the level of ApoAI in Caco-2 cells, the gene and protein expression of this protein was evaluated by Real-Time PCR and Western blot
An increase of paraoxonase 1 (PON1) gene and protein expression, and its secretion was observed in ApoAIp compared to CRISPR activation plasmid (Cp) cells (1.30 ± 0.08 versus 1.00 ± 0.05, p < 0.05, 1.70 ± 0.13 versus 1.00 ± 0.08, p < 0.001, and 2.48 ± 0.35 versus 1.00 ± 0.03, respectively, p < 0.01)
Summary
Introduction published maps and institutional affilCardiovascular diseases (CVD) represent the clinical manifestation of atherosclerosis and are a major threat to public health worldwide, with high prevalence and mortality rates [1,2]. Endothelial cells (EC) form the inner layer of cells of the arterial wall, and mediate the transport of molecules between plasma and tissues, regulate vascular tone, and synthesize and secrete a variety of factors that control lipid homeostasis, signal transduction, immunity, and inflammation [5]. Due to their location, EC are the first cells of the vessel wall exposed to injurious factors from the blood such as increased lipids and iations EC are the first cells of the vessel wall exposed to injurious factors from the blood such as increased lipids and iations.
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