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4-Hydroxynonenal (HNE) mediates oxidative stress-linked pathological processes; however, its role in the generation of reactive oxygen species (ROS) in macrophages is still unclear. Thus, this study investigated the sources and mechanisms of ROS generation in macrophages stimulated with HNE. Exposure of J774A.1 cells to HNE showed an increased production of ROS, which was attenuated by NADPH oxidase as well as 5-lipoxygenase (5-LO) inhibitors. Linked to these results, HNE increased membrane translocation of p47phox promoting NADPH oxidase activity, which was attenuated in peritoneal macrophages from 5-LO-deficient mice as well as in J774A.1 cells treated with a 5-LO inhibitor, MK886 or 5-LO siRNA. In contrast, HNE-enhanced 5-LO activity was not affected by inhibition of NADPH oxidase. Furthermore, leukotriene B(4), 5-LO metabolite, was found to enhance NADPH oxidase activity in macrophages. Altogether, these results suggest that 5-LO plays a critical role in HNE-induced ROS generation in murine macrophages through activation of NADPH oxidase.

Exaggerated levels of 4-hydroxynonenal (HNE) and 5-lipoxygenase (5-LO) co-exist in macrophages in atherosclerotic lesions, and activated macrophages produce MMP-9 that degrades atherosclerotic plaque constituents. This study investigated the effects of HNE on MMP-9 production, and the potential role for 5-LO derivatives in MMP-9 production in murine macrophages. Stimulation of J774A.1 cells with HNE led to activation of 5-LO, as measured by leukotriene B(4) (LTB(4)) production. This was associated with an increased production of MMP-9, which was blunted by inhibition of 5-LO with MK886, a 5-LO inhibitor or with 5-LO siRNA. A cysteinyl-LT(1) (cysLT(1)) receptor antagonist, REV-5901 as well as a BLT(1) receptor antagonist, U-75302, also attenuated MMP-9 production induced by HNE. Furthermore, LTB(4) and cysLT (LTC(4) and LTD(4)) enhanced MMP-9 production in macrophages, suggesting a pivotal role for 5-LO in HNE-mediated production of MMP-9. Among the MAPK pathways, LTB(4) and cysLT enhanced phosphorylation of ERK and p38 MAPK, but not JNK. Linked to these results, a p38 MAPK inhibitor as well as an ERK inhibitor blunted MMP-9 production induced by LT. Collectively, these data suggest that 5-LO-derived LT mediates HNE-induced MMP-9 production via activation of ERK and p38 MAPK pathways, consequently leading to plaque instability in atherosclerosis.

4-Hydroxynonenal (HNE) is known to be atherogenic, but its mechanism of action in atherogenesis is not clear. Therefore, this study investigated the role of HNE in macrophage foam cell formation and the underlying mechanism involved in HNE-induced expression of scavenger receptors (SRs). In the aortic sinus of ApoE-deficient mice fed a high-fat diet, multiple plaque lesions were accompanied by increased accumulation of HNE adducts in the enhanced Mac-2 stained area. In an in vitro study, HNE exposure to J774A.1 macrophages led to increased expression of class A SR (SR-A) and CD36 at the protein level with a concomitant increase in endocytic uptake of oxLDL. In contrast to CD36 protein expression, which was associated with an increase in mRNA expression, the HNE-enhanced SR-A protein expression was neither accompanied by its mRNA expression nor affected by actinomycin D. HNE enhanced the incorporation rates of 35S-Met/Cys into SR-A, and HNE-induced SR-A protein expression was effectively attenuated by translation inhibitors such as cycloheximide and rapamycin. Taken together, these data suggest that HNE contributes to macrophage foam cell formation through increased synthesis of SR-A at the level of mRNA translation, consequently leading to the progression of atherosclerosis.

5-Lipoxygenase (5-LO) has been suggested as a modulator of atherosclerotic plaque instability, however, its role in MMP production in vascular smooth muscle cells (VSMC) is still unclear. Thus, this study investigated the role of 5-LO in HNE-enhanced MMP-2 production in VSMC, and the mechanisms by which this enzyme could be activated by HNE. VSMC stimulated with HNE (1μM) produced MMP-2, which was markedly attenuated in 5-LO-deficient VSMC as well as in cells pretreated with a FLAP inhibitor, MK886, confirming a role for 5-LO metabolites in HNE-enhanced MMP-2 production. Related to these results, HNE increased nuclear translocation of 5-LO promoting 5-LO activity, which was attenuated not only by SB203580, a p38 MAPK inhibitor, but also by PD98059, an ERK inhibitor. In parallel, phosphorylation of p38 MAPK and ERK occurred as early as 15min after exposure to HNE, suggesting a potential role for p38 MAPK and ERK pathways in HNE-induced activation of 5-LO. Among leukotriene (LT) receptor antagonists, U-75302, a BLT receptor antagonist, but not MK-571 and Rev-5901, cysLT receptor antagonists, showed an inhibitory effect on HNE-enhanced MMP-2 production. Moreover, MMP-2 production in VSMC was also significantly increased by LTB 4 , but not by LTC 4 and LTD 4 . Collectively, these data suggest that 5-LO mediates HNE-enhanced MMP-2 production via LTB 4 -BLT receptor pathways, consequently leading to atherosclerotic plaque instability.

Increased levels of 4-hydroxynonenal (HNE) and 5-lipoxygenase (5-LO) coexist in atherosclerotic lesions but their relationship in atherogenesis is unclear. This study investigated the role of 5-LO in HNE-induced CD36 expression and macrophage foam cell formation, and the link between HNE and 5-LO. In J774A.1 murine macrophages, HNE (10 μM) enhanced CD36 expression in association with an increased uptake of oxLDL, which was blunted by inhibition of 5-LO with MK886, a 5-LO inhibitor, or with 5-LO siRNA. In peritoneal macrophages from 5-LO-deficient mice, HNE-induced CD36 expression was markedly attenuated, confirming a pivotal role of 5-LO in HNE-induced CD36 expression. In an assay for 5-LO activity, stimulation of macrophages with HNE led to increased leukotriene B4 production in the presence of exogenous arachidonic acid in association with an increased association of 5-LO to the nuclear membrane. Among the mitogen-activated protein kinase (MAPK) pathways involved in 5-LO phosphorylation, HNE predominantly activated p38 MAPK in macrophages, and the p38 MAPK inhibitor SB203580, but not an extracellular signal-regulated kinase inhibitor, suppressed HNE-induced LTB4 production. Collectively, these data suggest that p38 MAPK-mediated activation of 5-LO by HNE might enhance CD36 expression, consequently leading to the formation of macrophage foam cells.

4-Hydroxynonenal (HNE) accumulates at atherosclerotic lesions, but its role in the progression of atherosclerosis is not clear. Considering the role of matrix metalloproteinases (MMP) in plaque destabilization, we investigated the mechanism by which HNE induces MMP production in vascular smooth muscle cells (VSMC). VSMC stimulated by HNE (1.0 microM) produced enzymatically active MMP-2 with an increased promoter activity, which was abolished by mutation of the NF-kappaB binding site in the promoter region. The increased NF-kappaB activity with subsequent MMP-2 production by HNE was significantly attenuated by transfection with Akt siRNA as well as by pretreatment with the PI3K/Akt inhibitors LY294002 (10 microM) and SH-5 (1.0 microM). The phosphorylation of Akt occurred as early as 5 min in VSMC exposed to HNE and was markedly attenuated by inhibition of mitochondrial reactive oxygen species (ROS). Furthermore, the impact of mitochondrial ROS on HNE-induced Akt phosphorylation with subsequent MMP-2 production was also demonstrated in mitochondrial function-deficient VSMC, as well as in cells transfected with manganese superoxide dismutase. Taken together, these results suggest that HNE enhances MMP-2 production in VSMC via mitochondrial ROS-mediated activation of the Akt/NF-kappaB signaling pathways.