Noncanonical function of long myosin light chain kinase in increasing ER-PM junctions and augmentation of SOCE.

Increased endothelial permeability leads to excessive exudation of plasma proteins and leukocytes in the interstitium, which characterizes several vascular diseases including acute lung injury. The myosin light chain kinase long (MYLK-L) isoform is canonically known to regulate the endothelial permeability by phosphorylating myosin light chain (MLC-P). Compared to the short MYLK isoform, MYLK-L contains an additional stretch of ~919 amino acid at the N-terminus of unknown function. We show that thapsigargin and thrombin-induced SOCE was markedly reduced in Mylk-L-/- endothelial cells (EC) or MYLK-L-depleted human EC. These agonists also failed to increase endothelial permeability in MYLK-L-depleted EC and Mylk-L-/- lungs, thus demonstrating the novel role of MYLK-L-induced SOCE in increasing vascular permeability. MYLK-L augmented SOCE by increasing endoplasmic reticulum (ER)-plasma membrane (PM) junctions and STIM1 translocation to these junctions. Transduction of N-MYLK domain (amino acids 1-919 devoid of catalytic activity) into Mylk-L-/- EC rescued SOCE to the level seen in control EC in a STIM1-dependent manner. N-MYLK-induced SOCE augmented endothelial permeability without MLC-P via an actin-binding motif, DVRGLL. Liposomal-mediated delivery of N-MYLK mutant but not ∆DVRGLL-N-MYLK mutant in Mylk-L-/- mice rescued vascular permeability increase in response to endotoxin, indicating that targeting of DVRGLL motif within MYLK-L may limit SOCE-induced vascular hyperpermeability.

Neural Wiskott-Aldrich syndrome protein (N-WASP)-mediated p120-catenin interaction with Arp2-Actin complex stabilizes endothelial adherens junctions.

Stable adherens junctions (AJs) are required for formation of restrictive endothelial barrier. Vascular endothelial cadherin from contiguous endothelial cells forms AJs, which are stabilized intracellularly by binding of p120-catenin and cortical actin. Mechanisms inducing cortical actin formation and enabling its linkage with p120-catenin remain enigmatic. We altered the function of neural Wiskott-Aldrich syndrome protein (N-WASP), which induces actin polymerization through actin-related protein 2/3 complex (Arp2/3), to address the role of N-WASP in regulating AJ stability and thereby endothelial permeability. We show that depletion of N-WASP in endothelial cells impaired AJ adhesion and favored the organization of actin from cortical actin to stress fibers, resulting thereby in formation of leaky endothelial barrier. Exposure of the N-WASP-depleted endothelial cell monolayer to the permeability-increasing mediator, thrombin, exaggerated AJ disruption and stress fiber formation, leading to an irreversible increase in endothelial permeability. We show that N-WASP binds p120-catenin through its verprolin cofilin acid (VCA) domain, induces cortical actin formation through Arp2, and links p120-catenin with cortical actin. The interaction of N-WASP with p120-catenin, actin, and Arp2 requires phosphorylation of N-WASP at the Tyr-256 residue by focal adhesion kinase. Expression of the VCA domain of N-WASP or phosphomimicking (Y256D)-N-WASP mutant in endothelial cells stabilizes AJs and facilitates barrier recovery after thrombin stimulation. Our study demonstrates that N-WASP, by mediating p120-catenin interaction with actin-polymerizing machinery, maintains AJs and mitigates disruption of endothelial barrier function by edemagenic agents, therefore representing a novel target for preventing leaky endothelial barrier syndrome.

A new role for PTEN in regulating transient receptor potential canonical channel 6-mediated Ca2+ entry, endothelial permeability, and angiogenesis.

Phosphatase and tensin homologue (PTEN) is a dual lipid-protein phosphatase that catalyzes the conversion of phosphoinositol 3,4,5-triphosphate to phosphoinositol 4,5-bisphosphate and thereby inhibits PI3K-Akt-dependent cell proliferation, migration, and tumor vascularization. We have uncovered a previously unrecognized role for PTEN in regulating Ca(2+) entry through transient receptor potential canonical channel 6 (TRPC6) that does not require PTEN phosphatase activity. We show that PTEN tail-domain residues 394-403 permit PTEN to associate with TRPC6. The inflammatory mediator thrombin promotes this association. Deletion of PTEN residues 394-403 prevents TRPC6 cell surface expression and Ca(2+) entry. However, PTEN mutant, C124S, which lacks phosphatase activity, did not alter TRPC6 activity. Thrombin failed to increase endothelial monolayer permeability in the endothelial cells, transducing the Δ394-403 PTEN mutant. Paradoxically, we also show that thrombin failed to induce endothelial cell migration and tube formation in cells transducing the Δ394-403 PTEN mutant. Our results demonstrate that PTEN, through residues 394-403, serves as a scaffold for TRPC6, enabling cell surface expression of the channel. Ca(2+) entry through TRPC6 induces an increase in endothelial permeability and directly promotes angiogenesis. Thus, PTEN is indicated to play a role beyond suppressing PI3K signaling.

Activation of sphingosine kinase-1 reverses the increase in lung vascular permeability through sphingosine-1-phosphate receptor signaling in endothelial cells.

The lipid mediator sphingosine-1-phosphate (S1P), the product of sphingosine kinase (SPHK)-induced phosphorylation of sphingosine, is known to stabilize interendothelial junctions and prevent microvessel leakiness. Here, we investigated the role of SPHK1 activation in regulating the increase in pulmonary microvessel permeability induced by challenge of mice with lipopolysaccharide or thrombin ligation of protease-activating receptor (PAR)-1. Both lipopolysaccharide and thrombin increased mouse lung microvascular permeability and resulted in a delayed activation of SPHK1 that was coupled to the onset of restoration of permeability. In contrast to wild-type mice, Sphk1(-/-) mice showed markedly enhanced pulmonary edema formation in response to lipopolysaccharide and PAR-1 activation. Using endothelial cells challenged with thrombin concentration (50 nmol/L) that elicited a transient but reversible increase in endothelial permeability, we observed that increased SPHK1 activity and decreased intracellular S1P concentration preceded the onset of barrier recovery. Thus, we tested the hypothesis that released S1P in a paracrine manner activates its receptor S1P1 to restore the endothelial barrier. Knockdown of SPHK1 decreased basal S1P production and Rac1 activity but increased basal endothelial permeability. In SPHK1-depleted cells, PAR-1 activation failed to induce Rac1 activation but augmented RhoA activation and endothelial hyperpermeability response. Knockdown of S1P1 receptor in endothelial cells also enhanced the increase in endothelial permeability following PAR-1 activation. S1P treatment of Sphk1(-/-) lungs or SPHK1-deficient endothelial cells restored endothelial barrier function. Our results suggest the crucial role of activation of the SPHK1-->S1P-->S1P1 signaling pathway in response to inflammatory mediators in endothelial cells in regulating endothelial barrier homeostasis.

Role of protein tyrosine phosphatase 1B in vascular endothelial growth factor signaling and cell-cell adhesions in endothelial cells.

Vascular endothelial growth factor (VEGF) binding induces phosphorylation of VEGF receptor (VEGFR)2 in tyrosine, which is followed by disruption of VE-cadherin-mediated cell-cell contacts of endothelial cells (ECs), thereby stimulating EC proliferation and migration to promote angiogenesis. Tyrosine phosphorylation events are controlled by the balance of activation of protein tyrosine kinases and protein tyrosine phosphatases (PTPs). Little is known about the role of endogenous PTPs in VEGF signaling in ECs. In this study, we found that PTP1B expression and activity are markedly increased in mice hindlimb ischemia model of angiogenesis. In ECs, overexpression of PTP1B, but not catalytically inactive mutant PTP1B-C/S, inhibits VEGF-induced phosphorylation of VEGFR2 and extracellular signal-regulated kinase 1/2, as well as EC proliferation, whereas knockdown of PTP1B by small interfering RNA enhances these responses, suggesting that PTP1B negatively regulates VEGFR2 signaling in ECs. VEGF-induced p38 mitogen-activated protein kinase phosphorylation and EC migration are not affected by PTP1B overexpression or knockdown. In vivo dephosphorylation and cotransfection assays reveal that PTP1B binds to VEGFR2 cytoplasmic domain in vivo and directly dephosphorylates activated VEGFR2 immunoprecipitates from human umbilical vein endothelial cells. Overexpression of PTP1B stabilizes VE-cadherin-mediated cell-cell adhesions by reducing VE-cadherin tyrosine phosphorylation, whereas PTP1B small interfering RNA causes opposite effects with increasing endothelial permeability, as measured by transendothelial electric resistance. In summary, PTP1B negatively regulates VEGFR2 receptor activation via binding to the VEGFR2, as well as stabilizes cell-cell adhesions through reducing tyrosine phosphorylation of VE-cadherin. Induction of PTP1B by hindlimb ischemia may represent an important counterregulatory mechanism that blunts overactivation of VEGFR2 during angiogenesis in vivo.

Microtubule disassembly breaks down the barrier integrity of corneal endothelium.

Increased contractility of the peri-junctional actomyosin ring (PAMR) breaks down the barrier integrity of corneal endothelium. This study has examined the effects of microtubule disassembly on Myosin Light Chain (MLC) phosphorylation, a biochemical marker of actomyosin contraction, and barrier integrity in monolayers of cultured bovine corneal endothelial cells (BCEC). Exposure to nocodazole, which readily induced microtubule disassembly, led to disruption of the characteristically dense assembly of cortical actin cytoskeleton at the apical junctional complex (i.e., PAMR) and dispersion of ZO-1 from its normal locus. Nocodazole also led to an increase in phosphorylation of MLC. Concomitant with these changes, nocodazole caused an increase in permeability to HRP and FITC dextran (10 kDa) and a decrease in trans-endothelial electrical resistance (TER). Y-27632 (a Rho kinase inhibitor) and forskolin (known to inhibit activation of RhoA through direct elevation of cAMP) opposed the nocodazole-induced MLC phosphorylation, decrease in TER, and dispersion of ZO-1. Thrombin, which breaks down the barrier integrity of BCEC monolayers, also induced microtubule disassembly and MLC phosphorylation. Pre-treatment with paclitaxel to stabilize microtubules opposed the thrombin effects. These results suggest that microtubule disassembly breaks down the barrier integrity of BCEC through activation of RhoA and subsequent disruption of the PAMR. The thrombin effect also highlights that signaling downstream of GPCRs can also influence the organization of microtubules.

RhoGDI-1 modulation of the activity of monomeric RhoGTPase RhoA regulates endothelial barrier function in mouse lungs.

Rho family GTPases have been implicated in the regulation of endothelial permeability via their actions on actin cytoskeletal organization and integrity of interendothelial junctions. In cell culture studies, activation of RhoA disrupts interendothelial junctions and increases endothelial permeability, whereas activation of Rac1 and Cdc42 enhances endothelial barrier function by promoting the formation of restrictive junctions. The primary regulators of Rho proteins, guanine nucleotide dissociation inhibitors (GDIs), form a complex with the GDP-bound form of the Rho family of monomeric G proteins, and thus may serve as a nodal point regulating the activation state of RhoGTPases. In the present study, we addressed the in vivo role of RhoGDI-1 in regulating pulmonary microvascular permeability using RhoGDI-1(-/-) mice. We observed that basal endothelial permeability in lungs of RhoGDI-1(-/-) mice was 2-fold greater than wild-type mice. This was the result of opening of interendothelial junctions in lung microvessels which are normally sealed. The activity of RhoA (but not of Rac1 or Cdc42) was significantly increased in RhoGDI-1(-/-) lungs as well as in cultured endothelial cells on downregulation of RhoGDI-1 with siRNA, consistent with RhoGDI-1-mediated modulation RhoA activity. Thus, RhoGDI-1 by repressing RhoA activity regulates lung microvessel endothelial barrier function in vivo. In this regard, therapies augmenting endothelial RhoGDI-1 function may be beneficial in reestablishing the endothelial barrier and lung fluid balance in lung inflammatory diseases such as acute respiratory distress syndrome.

Oxidative lipidomics of apoptosis: redox catalytic interactions of cytochrome c with cardiolipin and phosphatidylserine.

The primary life-supporting function of cytochrome c (cyt c) is control of cellular energetic metabolism as a mobile shuttle in the electron transport chain of mitochondria. Recently, cyt c's equally important life-terminating function as a trigger and regulator of apoptosis was identified. This dreadful role is realized through the relocalization of mitochondrial cyt c to the cytoplasm where it interacts with Apaf-1 in forming apoptosomes and mediating caspase-9 activation. Although the presence of heme moiety of cyt c is essential for the latter function, cyt c's redox catalytic features are not required. Lately, two other essential functions of cyt c in apoptosis, that may rely heavily on its redox activity have been suggested. Both functions are directed toward oxidation of two negatively charged phospholipids, cardiolipin (CL) in the mitochondria and phosphatidylserine (PS) in the plasma membrane. In both cases, oxidized phospholipids seem to be essential for the transduction of two distinctive apoptotic signals: one is participation of oxidized CL in the formation of the mitochondrial permeability transition pore that facilitates release of cyt c into the cytosol and the other is the contribution of oxidized PS to the externalization and recognition of PS (and possibly oxidized PS) on the cell surface by specialized receptors of phagocytes. In this review, we present a new concept that cyt c actuates both of these oxidative roles through a uniform mechanism: its specific interactions with each of these phospholipids result in the conversion and activation of cyt c, transforming it from an innocuous electron transporter into a calamitous peroxidase capable of oxidizing the activating phospholipids. We also show that this new concept is compatible with a leading role for reactive oxygen species in the execution of the apoptotic program, with cyt c as the main executioner.

Cytochrome c release is required for phosphatidylserine peroxidation during Fas-triggered apoptosis in lung epithelial A549 cells.

Oxidation of phosphatidylserine (PtdSer) has been shown to play a pivotal role in signaling during cell apoptosis and subsequent recognition of apoptotic cells by phagocytes. However, the redox catalytic mechanisms involved in selective PtdSer oxidation during apoptosis remain poorly understood. Here we employed anti-Fas antibody CH-11-treated A549 cells as a physiologically relevant model to investigate the involvement of PtdSer oxidation and its potential mechanism during apoptosis. We demonstrated that ligation of CH-11 with its cognate receptor initiated execution of apoptotic program in interferon gamma-pretreated A549 cells as evidenced by activation of caspase and DNA fragmentation. A significant increase of cytochrome c (cyt c) content in the cytosol as early as 2 h after CH-11 exposure was detected indicating that Fas-induced apoptosis in A549 cells proceeds via extrinsic type II pathway and includes mitochondrial signaling. PtdSer was selectively oxidized 3 h after anti-Fas triggering while two more abundant phospholipids--phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn)--and the major intracellular antioxidant, glutathione, remained nonoxidized. A pan-caspase inhibitor, z-VAD, fully blocked cyt c release and oxidation of PtdSer in Fas-treated A549 cells. On the other hand, z-DQMD, a caspase-3 inhibitor, completely inhibited caspase-3 activity but did not fully block caspase-8 activation and release of cyt c. Importantly, z-DQMD failed to protect PtdSer from oxidation. In addition, in a model system, we demonstrated that peroxidase activity of cyt c was greatly enhanced in the presence of dioleoylphosphatidylserine containing liposomes by monitoring oxidation of 2',7'-dichlorodihydrofluorescein to 2',7'-dichlorofluorescein. We further showed that peroxidase activity of cyt c catalyzed oxidation of 1-palmitoyl-2-arachidonoyl-3-glycero-phosphoserine using a newly developed HPLC assay. MS analysis of 1-palmitoyl-2-arachidonoyl-3-glycero-phosphoserine revealed that in addition to its mono- and dihydroperoxides, several different PtdSer oxidation products can be formed. Overall, we concluded that cyt c acts as a catalyst of PtdSer oxidation during Fas-triggered A549 cell apoptosis.

Galphaq-TRPC6-mediated Ca2+ entry induces RhoA activation and resultant endothelial cell shape change in response to thrombin.

RhoA activation and increased intracellular Ca(2+) concentration mediated by the activation of transient receptor potential channels (TRPC) both contribute to the thrombin-induced increase in endothelial cell contraction, cell shape change, and consequently to the mechanism of increased endothelial permeability. Herein, we addressed the possibility that TRPC signals RhoA activation and thereby contributes in actinomyosin-mediated endothelial cell contraction and increased endothelial permeability. Transduction of a constitutively active Galphaq mutant in human pulmonary arterial endothelial cells induced RhoA activity. Preventing the increase in intracellular Ca2+ concentration by the inhibitor of Galphaq or phospholipase C and the Ca2+ chelator, BAPTA-AM, abrogated thrombin-induced RhoA activation. Depletion of extracellular Ca2+ also inhibited RhoA activation, indicating the requirement of Ca2+ entry in the response. RhoA activation could not be ascribed to storeoperated Ca2+ (SOC) entry because SOC entry induced with thapsigargin or small interfering RNA-mediated inhibition of TRPC1 expression, the predominant SOC channel in these endothelial cells, failed to alter RhoA activity. However, activation of receptor-operated Ca2+ entry by oleoyl-2-acetyl-sn-glycerol, the membrane permeable analogue of the Galphaq-phospholipase C product diacylglycerol, induced RhoA activity. Receptor-operated Ca2+ activation was mediated by TRPC6 because small interfering RNA-induced TRPC6 knockdown significantly reduced Ca2+ entry. TRPC6 knockdown also prevented RhoA activation, myosin light chain phosphorylation, and actin stress fiber formation as well as inter-endothelial junctional gap formation in response to either oleoyl-2-acetyl-sn-glycerol or thrombin. TRPC6-mediated RhoA activity was shown to be dependent on PKCalpha activation. Our results demonstrate that Galphaq activation of TRPC6 signals the activation of PKCalpha, and thereby induces RhoA activity and endothelial cell contraction.

Mechanisms of cardiolipin oxidation by cytochrome c: relevance to pro- and antiapoptotic functions of etoposide.

Execution of apoptotic program in mitochondria is associated with accumulation of cardiolipin peroxidation products required for the release of proapoptotic factors into the cytosol. This suggests that lipid antioxidants capable of inhibiting cardiolipin peroxidation may act as antiapoptotic agents. Etoposide, a widely used antitumor drug and a topoisomerase II inhibitor, is a prototypical inducer of apoptosis and, at the same time, an effective lipid radical scavenger and lipid antioxidant. Here, we demonstrate that cardiolipin oxidation during apoptosis is realized not via a random cardiolipin peroxidation mechanism but rather proceeds as a result of peroxidase reaction in a tight cytochrome c/cardiolipin complex that restrains interactions of etoposide with radical intermediates generated in the course of the reaction. Using low-temperature and ambient-temperature electron paramagnetic resonance spectroscopy of H(2)O(2)-induced protein-derived (tyrosyl) radicals and etoposide phenoxyl radicals, respectively, we established that cardiolipin peroxidation and etoposide oxidation by cytochrome c/cardiolipin complex takes place predominantly on protein-derived radicals of cytochrome c. We further show that etoposide can inhibit cytochrome c-catalyzed oxidation of cardiolipin competing with it as a peroxidase substrate. Peroxidase reaction of cytochrome c/cardiolipin complexes causes cross-linking and oligomerization of cytochrome c. With nonoxidizable tetraoleoyl-cardiolipin, the cross-linking occurs via dityrosine formation, whereas bifunctional lipid oxidation products generated from tetralinoleoyl-cardiolipin participate in the production of high molecular weight protein aggregates. Protein aggregation is effectively inhibited by etoposide. The inhibition of cardiolipin peroxidation by etoposide, however, is realized at far higher concentrations than those at which it induces apoptotic cell death. Thus, oxidation of cardiolipin by the cytochrome c/cardiolipin peroxidase complex, which is essential for apoptosis, is not inhibited by proapoptotic concentrations of the drug.

Cytochrome c acts as a cardiolipin oxygenase required for release of proapoptotic factors.

Programmed death (apoptosis) is turned on in damaged or unwanted cells to secure their clean and safe self-elimination. The initial apoptotic events are coordinated in mitochondria, whereby several proapoptotic factors, including cytochrome c, are released into the cytosol to trigger caspase cascades. The release mechanisms include interactions of B-cell/lymphoma 2 family proteins with a mitochondria-specific phospholipid, cardiolipin, to cause permeabilization of the outer mitochondrial membrane. Using oxidative lipidomics, we showed that cardiolipin is the only phospholipid in mitochondria that undergoes early oxidation during apoptosis. The oxidation is catalyzed by a cardiolipin-specific peroxidase activity of cardiolipin-bound cytochrome c. In a previously undescribed step in apoptosis, we showed that oxidized cardiolipin is required for the release of proapoptotic factors. These results provide insight into the role of reactive oxygen species in triggering the cell-death pathway and describe an early role for cytochrome c before caspase activation.

Effects of superoxide dismutase and polyclonal tumor necrosis factor-alpha antibodies on chloroacetate-induced cellular death and superoxide anion production by J774.A1 macrophages.

Dichloroacetate (DCA) and trichloroacetate (TCA) are by-products that are formed during the process of water chlorination and have been previously shown to induce superoxide anion (SA) production and cellular death when added to J774.A1 macrophage cultures. In this study, the effects of superoxide dismutase (SOD) and polyclonal tumor necrosis factor-alpha (TNF-alpha) antibodies on DCA- and TCA-induced SA production and cellular death have been tested on the J774.A1 macrophage cultures. TCA and DCA were added to different cultures either alone, each at a concentration of 16 mM, or in combination with SOD (2-12 units/ml), or with TNF-alpha antibodies (10 and 25 units/ml). Cells were incubated for 48 h, after which cellular death/viability, lactate dehydrognase (LDH) leakage by the cells, and SA production by the cells were determined. While TCA and DCA caused significant cellular toxicity, indicated by reduction in cellular viability and increases in LDH leakage and SA production, SOD addition resulted in significant reduction of the effects induced by the compounds. On the other hand, addition of TNF-alpha antibodies to the DCA- and TCA-treated cultures resulted in significant reduction of DCA- but not TCA-induced cellular death and SA production by the cells. Although these results suggest a significant role for SA in DCA- and TCA-induced cellular death, they may also suggest two different mechanisms for the chloroacetate-induced SA production by the cells.

Endogenously generated hydrogen peroxide is required for execution of melphalan-induced apoptosis as well as oxidation and externalization of phosphatidylserine.

Hydrogen peroxide (H(2)O(2)) is generated endogenously during execution of both intrinsic as well as extrinsic apoptotic programs suggesting that it may function as a secondary messenger in apoptotic pathways. In the present study, we investigated the role of endogenously generated H(2)O(2) by using two cell lines-HL-60 cells and its subclone, H(2)O(2) resistant HP100 cells overexpressing catalase (CAT). With the exception of CAT, we found no differences in the expression of other primary antioxidant enzymes (Cu/Zn-superoxide dismutase, Mn-superoxide dismutase, and glutathione peroxidase) or apoptosis-related proteins (Bcl-2 and Bax) in HP100 cells as compared with the parental HL-60 cells. Production of H(2)O(2) was readily detectable as early as 1 h after melphalan (Mel) exposure of HL-60 cells but not HP-100 cells. Biomarkers of apoptosis, such as release of cytochrome c, disruption of mitochondrial transmembrane potential, caspase-3 activation, and chromatin condensation, became apparent much later, 3 h and onward after Mel treatment of HL-60 cells. The emergence of essentially all biomarkers of apoptosis was dramatically delayed in HP100 cells as compared with HL-60 cells. A relatively minor phospholipid species, phosphatidylserine (PS), was markedly oxidized 3 h after Mel treatment in HL-60 cells (but not in HP100 cells) where it was significantly inhibited by exogenously added CAT. The two most abundant classes of membrane phospholipids, phosphatidylcholine and phosphatidyletanolamine, did not undergo any significant oxidation. PS oxidation took place 3 h after exposure of HL-60 cells to Mel and paralleled the appearance of cytochrome c in the cytosol. Neither cytochrome c release nor PS oxidation occurred in Mel-treated HP100 cells, indicating that both endogenous H(2)O(2) and cytochrome c were essential for selective PS oxidation detected in HL-60 cells. Mel-induced PS oxidation was also associated with externalization of PS on the surface of HL-60 cells. Given that 3-amino-1,2,4-triazole, a CAT inhibitor, suppressed the resistance of HP100 cells to apoptosis, production of reactive oxygen species, PS oxidation, and PS externalization induced by Mel, the results from the present study suggest that H(2)O(2) is critical for triggering the Mel-induced apoptotic program as well as PS oxidation and externalization.

Lipid antioxidant, etoposide, inhibits phosphatidylserine externalization and macrophage clearance of apoptotic cells by preventing phosphatidylserine oxidation.

Apoptosis is associated with the externalization of phosphatidylserine (PS) in the plasma membrane and subsequent recognition of PS by specific macrophage receptors. Selective oxidation of PS precedes its externalization/recognition and is essential for the PS-dependent engulfment of apoptotic cells. Because etoposide is a potent and selective lipid antioxidant that does not block thiol oxidation, we hypothesized that it may affect PS externalization/recognition without affecting other features of the apoptotic program. We demonstrate herein that etoposide induced apoptosis in HL-60 cells without the concomitant peroxidation of PS and other phospholipids. HL-60 cells also failed to externalize PS in response to etoposide treatment. In contrast, oxidant (H2O2)-induced apoptosis was accompanied by PS externalization and oxidation of different phospholipids, including PS. Etoposide potentiated H2O2-induced apoptosis but completely blocked H2O2-induced PS oxidation. Etoposide also inhibited PS externalization as well as phagocytosis of apoptotic cells by J774A.1 macrophages. Integration of exogenous PS or a mixture of PS with oxidized PS in etoposide-treated HL-60 cells reconstituted the recognition of these cells by macrophages. The current data demonstrate that lipid antioxidants, capable of preventing PS peroxidation, can block PS externalization and phagocytosis of apoptotic cells by macrophages and hence dissociate PS-dependent signaling from the final common pathway for apoptosis.