We show that an exogenous oxidized phospholipid is cytotoxic because it is readily internalized, migrates to mitochondria and then damages mitochondria in a way that activates the intrinsic caspase cascade. The sequence of events subsequent to internalization of the oxidation product included association with mitochondria, loss of mitochondrial sequestration of pro-apoptotic proteins, formation of the apoptosome with caspase-9 activation, and then caspase-3 activation. Activation of these caspases was required for HAzPC-induced cell death because irreversibly inhibiting caspase-9 suppressed cell death. Cellular mitochondria were the targets of the internalized HAzPC because over-expression of Bcl-XL suppressed cell death. The phospholipid itself was the pro-apoptotic agent because hydrolysis by PAF acetylhydrolase suppressed apoptosis.
We tested several cell lines, and circulating human cells (not shown) and found that each was susceptible to HAzPC-induced cell death, although the concentration of HAzPC required to induce death of these cells varied several fold, perhaps reflecting varied rates of internalization or hydrolysis. However, for each cell type HAzPC at low micromolar concentrations induced phosphatidylserine exposure, cytoplasmic accumulation of cytochrome C, and redistribution of AIF to the cytoplasm and nucleus.
AIF is released from its association with the inner mitochondrial membrane by apoptotic stimuli (26
) allowing it to migrate to the nucleus to cause nuclear condensation and cell death (28
). Cytosolic cytochrome C is an essential component of the apoptosome (29
) that cleaves pro-caspase 9 to its active form. The peptide zLEHD-fmk is a selective caspase-9 substrate and irreversible inhibitor that prevents cleavage of down stream targets such as caspase 3. Cells treated with HAzPC contained activated caspase 9, identified by fluorescent adduct derived from a fluorogenic irreversible inhibitor, and the cells contained active caspase 3. Activation of the executioner caspase 3 depended on caspase 9 cleavage because proteolytically activated caspase 3 fragments did not appear in cells containing irreversibly inactivated caspase 9. This inhibition of caspase activity also suppressed HAzPC-induced cell death, so HAzPC initiates death only via the apoptotic intrinsic caspase cascade.
homolog of C16
HAzPC, octadecyl azelaoyl phosphatidylcholine, previously has been identified as a lytic agent present in oxidized egg yolk phospholipid when erythrocytes were the target cells (30
). However, we find in nucleated cells that contain mitochondria that the primary effect of exogenous HAzPC was not lysis. The cells remained morphologically unremarkable and their plasma membrane presented a functional permeability barrier that retained cytoplasmic dyes. We did not observe (not shown) a prolonged influx of extracellular Ca++
, as found in human umbilical vein endothelial cells exposed to lysophosphatidylcholine (31
), or non-selective disruption of the plasma membrane as suggested for other short chain phospholipids (32
). Instead, we identified a marked change in cellular mitochondrial function in the absence of frank plasma membrane damage.
We conclude that the oxidized phospholipid HAzPC is pro-apoptotic because of its effect on intracellular mitochondria for several reasons. First, there is a temporal correlation between early mitochondrial dysfunction (3 to 4 h) and subsequent phosphatidylserine exposure (staring at 6h). Second, internalized HAzPC was concentrated as an intact phospholipid by mitochondria. Third, Bcl-XL
, which physically interacts with the mitochondrial outer membrane to promote cell survival (33
), suppressed HAzPC-induced cell death. Mitochondria isolated from cells over-expressing Bcl-XL
released little cytochrome C, which, in turn, resulted in fewer cells expressing phosphatidylserine on their surface after being exposed to HAzPC. Since specifically protecting mitochondria with ectopic Bcl-XL
blocked HAzPC cytotoxicity, the primary target of internalized HAzPC for this event are mitochondria. We currently do not know whether mitochondria are particularly sensitive to HAzPC, whether compromising their integrity is more apparent than with other organelles, or whether the high concentration of HAzPC encountered by mitochondria account for the mitotoxicity. It is apparent, however, that this lipid does not act as an indiscriminant detergent.
As anticipated from experiments with intact cells, HAzPC had a direct effect on isolated mitochondria and rapidly induced swelling in a concentration-dependent fashion. HAzPC, with an sn-1 ether bond and a 9 carbon di-acidic fragment derived from linoleoyl/linolenoyl oxidation, was more effective in this than its palmitoyl homolog, or the 5-carbon glutaroyl analog derived from arachidonate oxidation. It also was more effective than the shorter arachidonoyl fragmentation products containing 4-carbon butyroyl residue, or PAF that contains the two-carbon acetyl residue. The phospholipase A2
hydrolytic product of HAzPC, 1-hexadecyl-sn
-glycero-3-phosphocholine (lysoPAF), was only mildly effective in altering mitochondrial volume. This means that catabolism of this particular oxidized phospholipid by phospholipase A2
activity reduces its mitotoxicity. This relationship for the relative effectiveness of precursor and hydrolytic product does not hold for the other less potent oxidized phospholipids. A recent report (34
) shows that a calcium independent phospholipase A2
localizes to mitochondria and protects cells from oxidative insult and apoptotic death, suggesting phospholipid products such as HAzPC generated in vivo
are more toxic than their hydrolyzed components.
Components of oxidized LDL have previously been found to promote mitochondrial dysfunction (35
) and caspase 3 activation (36
), although the relevant mechanism(s) remain incompletely defined. The oxidized LDL components palmitoyl glutaroyl phosphatidylcholine and palmitoyl oxovaleroyl phosphatidylcholine (oxidative fragments of palmitoyl arachidonoyl phosphatidylcholine (4
) that are 4 carbons shorter than HAzPC) also enhance caspase 3 activity, although it requires 50 μM of these shorter chained oxidized phospholipids to achieve a modest increase in caspase 3 activity (38
), or a 50% decrease in viability (39
). This is a larger concentration difference than their effects on mitochondrial swelling, but internalization and intracellular distribution may also differ for individual oxidized phospholipids (40
). Reactive non-phospholipid oxidation products, such as acrolein (41
), 4-hydroxhexenal (43
) or 15-deoxy-Δ12,14
), depolarize isolated mitochondria and induce apoptosis. However, these chemically reactive lipids are unlikely to accumulate to a level able to affect mitochondrial function nor are they the cytotoxic agents destroyed by phospholipase A2
) or PAF acetylhydrolase (45
Oxidized LDL levels in carotid plaque are some 70-fold greater than circulating levels (50
), and accumulation of apoptotic macrophages and smooth muscle cells parallels the progression of atherosclerosis (6
). An important component of the toxic soup of atherosclerotic plaque are oxidized phospholipids because phospholipid hydrolysis reduces oxidized LDL cytotoxicty, at least to endothelial cells (10
). Linoleoyl (C18:2
) and linolenoyl (C18:3
) are the most abundant esterified polyunsaturated fatty acids, and both contain a 9,10 double bond. Preferential bond scission at this site (14
) creates phospholipids with a 9-carbon sn
-2 fragment that contain an aldehyde, which also is pro-apoptotic (51
), or the more oxidized azelaoyl residue. The azelaoyl residue is not a product of fatty acid β-oxidation, and has long served as a marker of chemical oxidation. Here we show phospholipids with this shortened fatty acyl residue not only are markers of oxidative processes, they directly participate in cell death by targeting mitochondria to initiate the intrinsic apoptotic caspase cascade.