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 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.

A Current Update on the Role of HDL-Based Nanomedicine in Targeting Macrophages in Cardiovascular Disease

“…It also enhances macrophage cholesterol efflux via ATP-binding cassette 1 (ABCA-1), and its deficiency leads to atherosclerotic complications [ 101 , 102 ]. In a recent report, the selective expression of ApoA-I and PON-1 has been shown to alleviate TNF-α induced endothelial dysfunction by reducing inflammatory and oxidative stress [ 103 ]. PON is recognized as a labile enzyme, which is quickly broken down and loses its function under specific circumstances.…”

Modulatory Effect of Lifestyle-Related, Environmental and Genetic Factors on Paraoxonase-1 Activity: A Review

“…Additionally, clarification of these mechanisms would be essential for potential therapeutic applications of the enzyme being developed in the future and for efficient disease management. For example, a novel therapeutic strategy based on CRISPR/Cas9 technology in order to reverse atherosclerotic processes through the transcriptional activation of endogenous PON1 and apolipoprotein-A1 (apo-A1) and thereby improve the HDL protective function has recently been evaluated on Caco-2 enterocytes [9]. The study gave promising results and is believed to bear potential for clinical application as the upregulation and subsequent overexpression of PON1 provides protection from oxidative and inflammatory stress and might in the future be an alternative therapy enabling to circumvent often inconvenient side effects of conventional pharmaceuticals used today as a standard.…”