Induction of ATF3 Gene Network by Triglyceride-Rich Lipoprotein Lipolysis Products Increases Vascular Apoptosis and Inflammation

Abstract

Objective Elevation of triglyceride-rich lipoproteins (TGRL) contributes to the risk for atherosclerotic cardiovascular disease (ASCVD). Our work has shown that TGRL lipolysis products in high physiological to pathophysiological concentrations cause endothelial cell injury; however, the mechanisms remain to be delineated. Approach and Results We analyzed the transcriptional signaling networks in arterial endothelial cells exposed to TGRL lipolysis products. When human aortic endothelial cells (HAEC) in culture were exposed to TGRL lipolysis products, activating transcription factor 3 (ATF3) was identified as a principal response gene. Induction of ATF3 mRNA and protein was confirmed by qRT-PCR and western blot. Immunofluorescence analysis showed that ATF3 accumulated in the nuclei of cells treated with lipolysis products. Nuclear expression of p-JNK, previously shown to be an initiator of the ATF3 signaling cascade, also was demonstrated. siRNA-mediated inhibition of ATF3 blocked lipolysis products-induced transcription of E-selectin and IL-8, but not IL-6 or NFκB. c-Jun, a downstream protein in the JNK pathway was phosphorylated while expression of NFκB-dependant JunB was down-regulated. Additionally, JNK siRNA suppressed ATF3 and p-c-Jun protein expression suggesting that JNK is up-stream of the ATF3 signaling pathway. In vivo studies demonstrated that infusion of TGRL lipolysis products into wild type mice induced nuclear ATF3 accumulation in carotid artery endothelium. ATF3−/− mice were resistant to vascular apoptosis precipitated by treatment with TGRL lipolysis products. Also peripheral blood monocytes isolated from postprandial humans had increased ATF3 expression as compared to fasting monocytes. Conclusion This study demonstrates that TGRL lipolysis products activate ATF3-JNK transcription factor networks and induce endothelial cells inflammatory response.

Lipotoxic brain microvascular injury is mediated by activating transcription factor 3-dependent inflammatory and oxidative stress pathways

“…Our previous studies indicated acute exposure to hydrolyzed TGRLs (TGRL lipolysis products) lead to inflammatory injury in human aortic endothelial cells (22,23,27,28), and we hypothesized similar effects may occur in the vasculature of the brain. In our in vitro model of cultured HBMECs, TEM revealed ultrastructural abnormalities in response to TGRL lipolysis products indicative of lipotoxic injury, including mitochondrial swelling and increased levels of lipid droplets in close proximity to mitochondria at 3 h. To further investigate the particular pathway responsible for this injury, we assayed for production of ROS and detected notably elevated levels of superoxide within 15 min of TGRL lipolysis product treatment that were significantly reduced by concomitant treatment with SOD, suggesting that TGRL lipolysis product-mediated HBMEC injury is dependent, at least in part, on elevated ROS.…”

“…by guest, on May 12, 2018 www.jlr.org Downloaded from (22), we investigated the involvement of ATF3 in the brain microvascular endothelial cell responses to TGRL lipolysis products. We observed markedly upregulated ATF3 expression within 3 h of treatment, as well as increased nuclear accumulation of ATF3.…”

“…Previous studies have shown that TGRL lipolysis products cause aortic endothelial injury, including inflammation, elevated expression of reactive oxygen species (ROS), and increased vascular permeability (22)(23)(24)(25)(26)(27)(28)(29). Additionally, activating transcription factor (ATF) 3 appears to play an important role as a master regulator of inflammation in the aortic endothelium in response to TGRL lipolysis products (22,30). However, the effects of these lipids on brain microvascular endothelial cells of the cerebrovasculature, and their potential involvement in lipotoxic dysfunction, are unknown and have not been comprehensively studied.…”

“…Quantitative RT-PCR (qRT-PCR) with SYBR as fluorescent reporter was used to quantify the expression of selected genes identified by GeneChip analysis. Specific human primers were designed with Primer Express 1.0 software (Applied Biosystems) using the gene sequences obtained from previously published Affymetrix Probeset IDs (Table 1) (22). Reactions were carried out in 384-well optical plates containing 25 ng RNA in each well.…”

“…To determine the extent of ATF3 nuclear accumulation in response to TGRL lipolysis products, HBMECs were grown to confluence on 12 mm round glass coverslips coated with attachment factor, placed in 24-well medical-grade polystyrene plates, and treated (n = 4 coverslips per treatment group) with media, TGRLs, TGRL lipolysis products, or LPL for 3 h. After treatment, HBMECs were fixed with 4% paraformaldehyde in PBS for 30 min at room temperature and then washed 3× with PBS. Cells were then permeabilized, blocked, and incubated with rabbit polyclonal anti-ATF3 (1:100 dilution, catalog number sc-188, Santa Cruz Biotechnology) as previously described (22). Subsequently, cells were treated with goat anti-rabbit secondary antibody conjugated to Alexa Fluor 488 (Molecular Probes), and the nuclei were counterstained for 5 min with DAPI (1 g/ml).…”