Glycoxydation Promotes Vascular Damage Via MAPK-ERK/JNK Pathways FILOMENA DE NIGRIS, 1 MONICA RIENZO, 1 MARCELLA SESSA, 1 TERESA INFANTE, 2 ELENA CESARIO, 1 LOUIS J. IGNARRO, 3 MOHAMMED AL-OMRAN, 4 ANTONIO GIORDANO, 5 WULF PALINSKI, 6 AND CLAUDIO NAPOLI 1,2 * 1 Department of General Pathology, U.O.C. Immunohematology, and Excellence Research Centre on Cardiovascular Disease, 1st School of Medicine, Second University of Naples, Naples, Italy 2 Fondazione Studio Diagnostica Nucleare (SDN), IRCCS, Naples, Italy 3 Department of Pharmacology, University of California at Los Angeles, Los Angeles, California 4 Peripheral Vascular Disease Research Chair, College of Medicine, King Saud University, Riyadh, Saudi Arabia 5 Department of Biology, Temple University, Philadelphia, Pennsylvania 6 Department of Medicine, University of California San Diego, La Jolla, California Oxidation and glycation enhance foam cell formation via MAPK/JNK in euglycemic and diabetic subjects. Here, we investigated the effects of glycated and oxidized LDL (glc-oxLDL) on MAPK-ERK and JNK signaling pathways using human coronary smooth muscle cells. Glc-oxLDL induced a broad cascade of MAPK/JNK-dependent signaling transduction pathways and the AP-1 complex. In glc-oxLDL treated coronary arterioles, tumor necrosis factor (TNF) a increased JNK phosphorylation, whereas protein kinase inhibitor dimethylaminopurine (DMAP) prevented the TNF-induced increase in JNK phosphorylation. The role of MKK4 and JNK were then investigated in vivo, using apolipoprotein E knockout (ApoE À/À ) mice. Peritoneal macrophages, isolated from spontaneously hyperlipidemic but euglycemic mice showed increases in both proteins and phosphorylated proteins. Compared to streptozotocin-treated diabetic C57BL6 and nondiabetic C57BL6 Wt mice, in streptozotocin-diabetic ApoE À/À mice, the increment of foam cell formation corresponded to an increment of phosphorylation of JNK1, JNK2, and MMK4. Thus, we provide a first line of evidence that MAPK-ERK/JNK pathways are involved in vascular damage induced by glycoxidation. J. Cell. Physiol. 227: 3639–3647, 2012. ß 2012 Wiley Periodicals, Inc. Vascular damage is promoted by complex interactions between modified lipoproteins, monocyte-derived macrophages that become foam cells, T lymphocytes, and smooth muscle cells in, or derived from, the arterial media (Prieur et al., 2010; Libby et al., 2011). c-Jun N-terminal kinases (JNKs) belong to the mitogen-activated protein kinase (MAPK) family (Choi et al., 2008), and are implicated in most of the cellular processes involved in the development of atherosclerosis (Choi et al., 2008; Muslin, 2008). Ten JNK isoforms have been identified in the human brain, corresponding to alternative spliced isoforms derived from the JNK1, JNK2, and JNK3 genes (Hahn et al., 2011). JNK1 and JNK2 are widely expressed. In contrast, JNK3 has a more limited pattern of expression that is largely restricted to brain, heart, and testis (Bogoyevitch, 2006). Although mice lacking JNK1 or JNK2 appear morphologically normal, they are immune-compromised because of T-cell defects (Junyent et al., 2011). Recent studies in murine models have shown that JNK1 regulates insulin resistance, obesity and seems to be protective against brief ischemia during acute myocardial infarction (Wei et al., 2011; Xie et al., 2011). Whereas JNK2 is required for collagen induced arthritis (Denninger et al., 2011). Several experimental and clinical studies have demonstrated that oxidized low-density lipoprotein (oxLDL) is central in the pathogenesis of vascular dysfunction and atherogenesis (Napoli et al., 1997a, 1997b, 2003; Sun and Chen, 2011). Indeed, oxidized low-density lipoprotein is already prevalent in atherosclerotic lesions of human fetuses and children (Napoli et al., 1997b, 1999, 2001; Steinberg and Witztum, 2010; Mazie `re et al., 2011). In diabetic subjects, proteins exposed to elevated glucose concentration Abbreviations: AGE, advanced glycation end products; AP-1 complex, activator protein 1 complex; EMSA, electrophoretic mobility shift assay; ERK-1, extracellular regulated kinase 1; ERK-2, extracellular regulated kinase 2; Fra-1, fos related antigen 1; Fra-2, fos related antigen 2; glcLDL, glycosylated low-density lipoprotein; glc-oxLDL, glycosylated and oxidized low-density lipoprotein; JNK, Jun kinase; LDL, low-density lipoprotein; MAPK, mitogen-activated protein kinase; MDA, malondialdehyde; oxLDL, oxidized low- density lipoprotein; PD98059, MAPK synthetic inhibitor; RAGE, advanced glycation end product receptor; SAPK, stress-activated protein kinase; TBARS, thiobarbituric acid reactive substances; VSMC, vascular smooth muscle cells. Conflicts of interests: The authors have no conflicts of interests in the connection of this study. Contract grant sponsor: PRIN 2008. Contract grant sponsor: Foundation Jerome Lejeune. Contract grant sponsor: NIH HL-089559. Contract grant sponsor: Ellisson Medical Foundation Award AG-SS 1851-07. *Correspondence to: Prof. Claudio Napoli, MD, PhD, MBEth, FACA, Chair of Clinical Pathology, Department of General Pathology, Second University of Naples, Via Costantinopoli, 16, 80138 Naples, Italy. E-mail: claudio.napoli@unina2.it Manuscript Received: 31 January 2012 Manuscript Accepted: 1 February 2012 Accepted manuscript online in Wiley Online Library (wileyonlinelibrary.com): 13 February 2012. DOI: 10.1002/jcp.24070 ORIGINAL RESEARCH ARTICLE 3639 Journal of Journal of Cellular Physiology Cellular Physiology ß 2012 WILEY PERIODICALS, INC.