Oxidized Phospholipids in Minimally Modified Low Density Lipoprotein Induce Apoptotic Signaling via Activation of Acid Sphingomyelinase in Arterial Smooth Muscle Cells

Abstract

Oxidized phospholipids, including 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC) and 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC), typically present in minimally modified low density lipoprotein, have been found in atherosclerotic lesions. These compounds are gaining increasing importance as inducers of different cellular responses (inflammation, proliferation, or cell death). It was the aim of this study to understand their impact on intracellular signal transduction pathways that are responsible for these biological effects. We found that in arterial smooth muscle cells, PGPC and POVPC activated sphingomyelinases, in particular the acid isoform, which is known to participate in the very early phase of apoptotic stress responses. In addition, mitogenactivated protein kinases, which are involved in induction of stress response and apoptosis were phosphorylated (activated). Finally, activation of caspase 3 was observed, showing that stimulation of smooth muscle cells with POVPC and PGPC is associated with apoptosis. Stimulation of all these enzymes by the oxidized phospholipids almost perfectly matched their activation by minimally modified LDL. Consequently, these phospholipids seem to be responsible for the effect of this particle on cell signaling. Survival and proliferation pathways including NF-B or AKT kinase were not induced by POVPC and PGPC. Experiments with a specific inhibitor of acid sphingomyelinase named NB6 showed that this enzyme plays a central role in mediating the apoptotic effects of the oxidized lipids. Thus, we conclude that modified phospholipids induce signaling cascades via activation of acid sphingomyelinase finally leading to apoptosis of smooth muscle cells, which is a detrimental process in the development of atherosclerosis.

Import and fate of fluorescent analogs of oxidized phospholipids in vascular smooth muscle cells

“…PGPC and POVPC have been reported to elicit apoptotic signaling in VSMCs (11). Issues that remain to be clarified include the identification of their primary molecular targets.…”

“…To address the question of whether the cellular distribution of the fluorescent lipid analogs represents the behavior of their unlabeled counterparts, at least in a qualitative manner, we determined their effect on the activation of acid sphingomyelinase, which is a key component of the apoptotic signaling induced by PGPC and POVPC (11). The data presented in Fig.…”

“…It has been demonstrated that the truncated sn-2 substituents of these compounds are major determinants of their bioactivity (9). Studies from several laboratories have revealed that the biological effects triggered by minimally modified LDL can largely be attributed to phospholipid oxidation products (7,8,10,11). Their increased levels in atherosclerotic plaques (12,13) and the increased antibody titers against oxidized phospholipids in humans and mice with lesions (14-16) attract attention to the pathological relevance of these molecules.…”

“…PGPC and POVPC exhibit proinflammatory actions (7,(17)(18)(19) but also increase the synthesis of protective enzymes such as heme oxygenase 1 (20). In human vascular smooth muscle cells (VSMCs), POVPC and, to a lesser extent, PGPC activated apoptotic signaling pathways via the stimulation of acid sphingomyelinase (11).…”

“…VSMCs can acquire a broad spectrum of different phenotypes in response to changes in a variety of physiological or pathological factors (21,22), including oxidized phospholipids (11,18,23). Nonetheless, the primary molecular targets and the mechanisms of their activation by these molecules are largely unknown.…”