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.

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Abstract-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 Ϫ/Ϫ 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 Ϫ/Ϫ lungs or SPHK1-deficient endothelial cells restored endothelial barrier function. Our results suggest the crucial role of activation of the SPHK13 S1P3 S1P1 signaling pathway in response to inflammatory mediators in endothelial cells in regulating endothelial barrier homeostasis. Key Words: sphingosine kinase Ⅲ lung vascular permeability Ⅲ thrombin Ⅲ PAR-1 Ⅲ RhoGTPases Ⅲ S1P1 Ⅲ S1P T he vascular endothelium forms a semipermeable barrier separating intravascular and tissue compartments. Disruption of endothelial barrier is a crucial factor in the pathogenesis of tissue inflammation, the hallmark of inflammatory diseases such as the acute respiratory distress syndrome. 1 Increased microvessel endothelial permeability leads to protein-rich alveolar edema that severely impairs oxygenation. 2 Thrombin, a serine protease, generated during sepsis and intravascular coagulation, ligates the endothelial cell surface receptor protease activating receptor 1 (PAR-1) and increases endothelial permeability. 1,[3][4][5][6] This increase in endothelial permeability is typically followed by a recovery period of Ϸ2 hours, during which barrier integrity is restored. 7,8 It has been surmised that PAR-1 signaling stimulates intrinsic repair mechanisms that restore barrier function. 7-9 Sphingosine-1-phosphate (S1P), a lipid mediator, was shown to be 1 such factor promoting endothelial barrier function. 10 -13 S1P binds to S...

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Abstract-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. (Circ Res. 2008;102:1182-1191.)Key Words: protein tyrosine phosphatase 1B Ⅲ vascular endothelial growth factor Ⅲ endothelial cell Ⅲ cell-cell adhesions Ⅲ angiogenesis

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 tetraoleoylcardiolipin, 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.Mitochondria play a central role in the execution of apoptotic program realized through intrinsic mechanisms and extrinsic pathways in type II cells (Scaffidi et al., 1998). It is well-documented that one of the early mitochondrial responses to proapoptotic stimuli is the generation of reactive oxygen species (ROS) (Raha and Robinson, 2001). Whereas the overall significance of ROS production in apoptosis has been established by its inhibition by different antioxidant enzymes and free radical scavengers (Nomura et al., 1999;Genova et al., 2003), specific ROS-dependent mechanisms of apoptosis are still elusive. We have demonstrated recently that cytochrome c acts as a redox catalyst in oxidizing a mitochondria-specific phospholipid, cardiolipin, thus facilitating the accumulation of cardiolipin hydroperoxides (CL-OOH) required for the release of proapoptotic factors from mitochondria into the cytosol . This suggests that free radical scave...

Abstract-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 Ϫ/Ϫ mice. We observed that basal endothelial permeability in lungs of RhoGDI-1 Ϫ/Ϫ 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 Ϫ/Ϫ 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. (Circ Res. 2007;101:50-58.)

Phosphatase and tensin homologue (PTEN) is a dual lipidprotein 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 ⌬394 -403 PTEN mutant. Paradoxically, we also show that thrombin failed to induce endothelial cell migration and tube formation in cells transducing the ⌬394 -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.Phosphatase and tensin homologue (PTEN), 2 a dual lipidprotein phosphatase, is composed of an N-terminal phosphatase domain, a C2 domain, and a C-terminal tail domain that has a PDZ (post-synaptic density protein (PSD95), Drosophila disc large tumor suppressor (DlgA), and zonula occludens-1 protein (ZO1)) domain binding sequence (1, 2). The phosphatase domain specifically dephosphorylates the D3 inositol head group of phosphoinositol 3,4,5-triphosphate (PIP 3 ) leading to generation of phosphoinositol 4,5-bisphosphate (PIP 2 ) (1, 2). Thus, through this domain PTEN negatively regulates PI3K-Akt-dependent signaling and thereby controls diverse cellular responses such as neointima formation, neutrophil migration and chemotaxis, angiogenesis, and tumor formation (3-9). PIP 2 is also the key source for generating cellular diacylglycerol (DAG) and inositol triphosphate, both of which increase intracellular Ca 2ϩ (10). DAG increases intracellular Ca 2ϩ by directly activating plasmalemmal Ca 2ϩ entry through receptor-operated Ca 2ϩ (ROC) channels (11-13). Inositol triphosphate mobilizes Ca 2ϩ from endoplasmic reticulum (ER) stores (11-14). Depletion of ER stores activates stromal interacting molecule 1 (STIM1), which induces Ca 2ϩ entry from store-operated Ca 2ϩ channels (15, 16). The domain structure of PTEN is consistent with the possibility that PTEN may have a role beyond acting as a phosphatase. In fact, PTEN has been shown to serve as a scaffold for MAGI (membrane-associated guanylate kinase) and Na ϩ /Hϩ exchanger regulatory factor (17)(18)(19)(20). This has been suggested to contribute to the stabilization of intercellular ju...