Journal
CIRCULATION RESEARCH
Volume 104, Issue 2, Pages 210-U147Publisher
LIPPINCOTT WILLIAMS & WILKINS
DOI: 10.1161/CIRCRESAHA.108.181040
Keywords
minimally oxidized LDL; reactive oxygen species; NADPH oxidase 2; RANTES; atherosclerosis
Funding
- National Core Research Center program [R15-2006-020-00000-0]
- The Ministry of Education, Science and Technology/Korea Science and Engineering Foundation
- Ewha Womans University
- Korea Health 21 RD [A06-00043579]
- Ministry of Health Welfare
- Seoul RD Program [10527]
- National Research Laboratory Program [ROA-2007-00020004-00]
- NIH [HL081862]
- American Heart Association [0530159N]
- National Research Foundation of Korea [2006-0051735, 2007-0056788, R31-2008-000-10010-0] Funding Source: Korea Institute of Science & Technology Information (KISTI), National Science & Technology Information Service (NTIS)
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Oxidative modification of low-density lipoprotein (LDL) plays a causative role in the development of atherosclerosis. In this study, we demonstrate that minimally oxidized LDL (mmLDL) stimulates intracellular reactive oxygen species (ROS) generation in macrophages through NADPH oxidase 2 (gp91phox/Nox2), which, in turn, induces production of RANTES and migration of smooth muscle cells. Peritoneal macrophages from gp91phox/Nox2(-/-) mice or J774 macrophages in which Nox2 was knocked down by small interfering RNA failed to generate ROS in response to mmLDL. Because mmLDL-induced cytoskeletal changes were dependent on Toll-like receptor (TLR)4, we analyzed ROS generation in peritoneal macrophages from wild-type, TLR4(-/-), or MyD88(-/-) mice and found that mmLDL-mediated ROS was generated in a TLR4-dependent, but MyD88-independent, manner. Furthermore, we found that ROS generation required the recruitment and activation of spleen tyrosine kinase (Syk) and that mmLDL also induced phospholipase PLC gamma 1 phosphorylation and protein kinase C membrane translocation. Importantly, the phospholipase C gamma 1 phosphorylation was reduced in J774 cells expressing Syk-specific short hairpin RNA. Nox2 modulated mmLDL activation of macrophages by regulating the expression of proinflammatory cytokines interleukin-1 beta, interleukin-6, and RANTES. We showed that purified RANTES was able to stimulate migration of mouse aortic smooth muscle cells and addition of neutralizing antibody against RANTES abolished the migration of mouse aortic smooth muscle cells stimulated by mmLDL-stimulated macrophages. These results suggest that mmLDL induces generation of ROS through sequential activation of TLR4, Syk, phospholipase C gamma 1, protein kinase C, and gp91phox/Nox2 and thereby stimulates expression of proinflammatory cytokines. These data help explain mechanisms by which endogenous ligands, such as mmLDL, can induce TLR4-dependent, proatherogenic activation of macrophages. (Circ Res. 2009; 104: 210-218.)
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