Original article Triglyceride-rich lipoproteins from hypertriglyceridemic subjects induce a pro-inflammatory response in the endothelium: Molecular mechanisms and gene expression studies G.D. Norata a,c , L. Grigore c , S. Raselli c , P.M. Seccomandi a , A. Hamsten b , F.M. Maggi a , P. Eriksson b , A.L. Catapano a,c, * a Department of Pharmacological Sciences, University of Milan, Via Balzaretti 9, 20133 Milan, Italy b Atherosclerosis Research Unit, King Gustaf V Research Institute, Department of Medicine, Karolinska Institute, Karolinska University Hospital, Stockholm, Sweden c Center for the Study of Atherosclerosis, Ospedale Bassini, Cinisello Balsamo, Italy Received 26 April 2005; received in revised form 25 January 2006; accepted 26 January 2006 Available online 06 March 2006 Abstract Triglyceride-rich lipoproteins (TGRLs) are a cardiovascular risk factor and induce endothelial dysfunction. In the present study we investi- gated the effects of TGRLs from type IV hyperlipidemic and normolipidemic subjects on endothelial activation focusing on the effects on intracellular pathways and gene expression. A total of 54 subjects, 30 hypertriglyceridemic (triglyceride (TG) levels 284 ± 101 mg/dl) and 23 normotriglyceridemic (TG levels 109 ± 40 mg/dl) were enrolled as lipoprotein donors. TGRLs were isolated from hypertriglyceridemic (H- TGRL) and normotriglyceridemic (N-TGRL) subjects. RNA from human endothelial cells incubated with N-TGRL or H-TGRL was prepared for cDNA microarray analyses. Western blotting was used to study intracellular signaling pathways. Regulated genes were further studied with real-time PCR, immunofluorescence and FACS. Furthermore, a protein/DNA array and chromatin-immunoprecipitation were used to identify transcription factors involved in the observed effects. Both N-TGRL and H-TGRL activated ERK1/2 and p38 MAPK. However, there were differences in the pattern of upregulated target genes between the two types of lipoproteins in HUVECs and/or HAECs: PAI-1, VCAM-1, ELAM-1 and MCP-1 were upregulated by both N-TGRL and H-TGRL, while PECAM-1, IL-6 and ADAMTs1 were selectively upregulated by H-TGRL. Chromatin immunoprecipitation analysis demonstrated the involvement of transcription factors NF-kB and CREB in the activation of these genes. These results support the possible involvement of hypertriglyceridemic TGRLs in endothelial dysfunction via induction of a pro- inflammatory and pro-thrombotic state. © 2006 Elsevier Ltd. All rights reserved. Keywords: Lipoproteins; Endothelial function; Gene expression; Signal transduction 1. Introduction Triglyceride-rich lipoproteins (TGRLs), including very low density lipoprotein (VLDLs), chylomicrons and their remnants, are regarded as risk factors for cardiovascular disease and have been shown to impair vasorelaxation [1,2]. Moreover, elevated fasting remnant lipoprotein levels are associated with impaired coronary vasomotor function [3]. TGRL remnant particles are independently associated with the presence, severity and pro- gression of atherosclerosis [4], and high fasting remnant lipo- protein levels predict carotid artery intimal thickness and future coronary events in subjects with coronary artery disease [5,6]. Human vascular endothelial function evaluated as brachial ar- tery flow-dependent dilatation is decreased after a fatty meal in healthy subjects, starting after 2 h, and is independent of total or low density lipoprotein (LDL) cholesterol levels [7,8]. The elevation in circulating free fatty acids (FFAs) impairs en- dothelium dependent vasodilation [9], and the impairment in endothelial function may be dependent on enhanced oxidative stress [10]. We have recently shown that VLDLs from normo- triglyceridemic subjects modulate the expression of genes in- volved in endothelial cell (EC) activation [11]. To date plasmi- nogen activator inhibitor-1 (PAI-1) is the only gene that has www.elsevier.com/locate/yjmcc Journal of Molecular and Cellular Cardiology 40 (2006) 484–494 * Corresponding author. Tel.: +39 02 5031 8302; fax: +39 02 5031 8386. E-mail address: Alberico.Catapano@unimi.it (A.L. Catapano). 0022-2828/$ - see front matter © 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.yjmcc.2006.01.022