Angiotensin II induces phenotype-dependent apoptosis in vascular smooth muscle cells.

Angiotensin II regulates vascular structure through growth and apoptosis, with implications in pathophysiology. Subtypes of vascular smooth muscle cells with specific morphology, growth, or apoptotic features have been isolated. Here, we investigated the effects of angiotensin II on apoptosis of 2 morphologically different rat aortic smooth muscle cell phenotypes. Spindle and epithelioid cell lines cultured under low serum conditions were stimulated by angiotensin II. Responsiveness was evaluated by calcium signaling. In both phenotypes, an angiotensin II type 1 receptor-mediated transient intracellular calcium peak arose from intracellular pools. However, a sustained nifedipine-sensitive calcium entry occurred specifically in epithelioid cells. Angiotensin II did not impair spindle cell survival, whereas a delayed reduction in cell number occurred in epithelioid cells. Cell death through apoptosis was characterized by cellular and nuclear morphology. Consistently, DNA fragmentation, evaluated by biochemical quantification, nuclei staining, and ladders, and caspase 3-like activity were promoted by angiotensin II in epithelioid cells. Kinetics of annexin V binding showed that apoptosis was a delayed process. Angiotensin II-induced apoptosis of epithelioid cells was prevented by angiotensin II type 1 but not type 2 receptor antagonists and was inhibited by a calcium chelator or calcium antagonist. Conversely, epithelioid cell apoptosis could be induced by a calcium ionophore. Thus, the death signaling promoted by angiotensin II in epithelioid cells involves type 1 receptor-mediated calcium entry. These data suggest that angiotensin II can promote angiotensin II type 1 receptor-mediated apoptosis in vascular smooth muscle cells, depending on their phenotype. This process may play a role in vascular remodeling in cardiovascular diseases.

Mildly oxidized LDL induces activation of platelet-derived growth factor beta-receptor pathway.

BACKGROUND: Mildly oxidized LDL (moxLDL) is thought to play a role in atherogenesis. MoxLDL induces derivatization of cell proteins and triggers a variety of intracellular signaling. We aimed to investigate whether moxLDL-induced protein derivatization may influence the activity of platelet-derived growth factor receptor beta (PDGFRbeta), a tyrosine kinase receptor of major importance in vascular biology and atherogenesis. METHODS AND RESULTS: In cultured rabbit arterial smooth muscle cells, moxLDL induces activation of the PDGFRbeta signaling pathway, as shown by PDGFRbeta tyrosine phosphorylation on Western blot and coimmunoprecipitation of SH2-containing proteins. The cellular events involved in the moxLDL-induced PDGFRbeta activation can be summarized as follows. Oxidized lipids from moxLDL trigger two phases of PDGFRbeta activation involving two separate mechanisms, as shown by experiments on cultured cells (in situ) and on immunopurified PDGFRbeta (in vitro): (1) the first phase may be mediated by 4-hydroxynonenal, which induces PDGFRbeta adduct formation and subsequent PDGFRbeta activation (antioxidant-insensitive step); (2) the second phase involves ceramide-mediated generation of H(2)O(2) (these steps being inhibited by tosylphenylalanylchloromethylketone, an inhibitor of ceramide formation, and by antioxidant BHT, exogenous catalase, or overexpressed human catalase). Because 4-hydroxynonenal-PDGFRbeta adducts are also detected in atherosclerotic aortas, it is suggested that this novel mechanism of moxLDL-induced PDGFRbeta activation may occur during atherogenesis. CONCLUSIONS: MoxLDL acts as a local autoparacrine mediator in the vascular wall, and PDGFRbeta acts as a sensor for both oxidized lipids and oxidative stress. This constitutes a novel mechanism of PDGFRbeta activation in atherosclerotic areas.

Oxidized LDLs alter the activity of the ubiquitin-proteasome pathway: potential role in oxidized LDL-induced apoptosis.

Oxidized low-density lipoproteins (oxLDL) play a role in the genesis of atherosclerosis. OxLDL are able to induce apoptosis of vascular cells, which is potentially involved in the formation of the necrotic center of atherosclerotic lesions, plaque rupture, and subsequent thrombotic events. Because oxLDL may induce structural modifications of cell protein and altered proteins may impair cell viability, the present work aimed to evaluate the extent of protein alterations, the degradation of modified proteins through the ubiquitin-proteasome system (a major degradative pathway for altered and oxidatively modified proteins) and their role during apoptosis induced by oxLDL. This paper reports the following: 1) oxLDL induce derivatization of cell proteins by 4-hydroxynonenal (4-HNE) and ubiquitination. 2) Toxic concentrations of oxLDL elicit a biphasic effect on proteasome activity. An early and transient activation of endogenous proteolysis is followed rapidly by a subsequent decay (resulting probably from the 26S proteasome inhibition) and followed later by the inhibition of the 20S proteasome (as assessed by inhibition of sLLVY-MCA hydrolysis). 3) Specific inhibitors of proteasome (lactacystin and proteasome inhibitor I) potentiated considerably the toxicity of oxLDL (nontoxic doses of oxLDL became severely toxic). The defect of the ubiquitination pathway (in temperature-sensitive mutants) also potentiated the toxicity of oxLDL. This suggests that the ubiquitin-proteasome pathway plays a role in the cellular defenses against oxLDL-induced toxicity. 4) Dinitrophenylhydrazine (DNPH), an aldehyde reagent, prevented both the oxLDL-induced derivatization of cell proteins and subsequent cytotoxicity. Altogether, the reported data suggest that both derivatization of cell proteins (by 4-HNE and other oxidized lipids) and inhibition of the proteasome pathway are involved in the mechanism of oxLDL-induced apoptosis.

The CBF.78 monoclonal antibody to human sialophorin has distinct properties giving new insights into the CD43 marker and its activation pathway.

We confirm here the CD43 specificity of the CBF.78 monoclonal antibody (mAb) and compare its phenotypic and functional capacities to classical group-A mAbs (DFT1, MEM-59) and to 2 other CD43 mAbs (RDP/AD9, 161-46). It reacts with stable human CD43 transfectants in a sialic acid independent way and blocks completely cell binding of RDP/AD9 or 161-46 more or less but not DFT1 and MEM-59. Its distribution differs from all other CD43. B lymphocytes, but surprisingly the majority of granulocytes or monocytes are CBF.78 negative. CBF.78 is expressed on all T lymphocytes, but the number of CBF.78 molecules/cell is low and equally represented on resting T CD4 and CD8 cells. In comparison to naive T lymphocytes, CD45RO cells increase their CBF.78 epitopes much more than other CD43 epitopes. At a single cell level, confocal microscopy shows that CBF.78 can exist independently of other epitopes. CBF.78 is able to induce homotypic adhesion in different cell lines but not in peripheral blood lymphocytes and is unable to relocalise the targeted molecules. U937 cell line that is not agglutinated by CBF.78 (or RDP/AD9) undergoes a stronger adhesion with PMA, when this reagent is combined with this mAb. By itself CBF.78 is unable to activate T lymphocytes and to costimulate CD3 mAbs but partially blocks PMA. The phosphorylation of the tyrosine kinase p59fyn and p56lck, driven by CBF.78, is weak and almost blocked by PMA. Altogether these data support the hypothesis that there are at least 3 modes of interaction between PKC and CD43 pathways: each pathway is inhibitory towards the other but the CD43 one can also be synergistic.

Bcl-2 alters the balance between apoptosis and necrosis, but does not prevent cell death induced by oxidized low density lipoproteins.

Oxidized low density lipoproteins (oxLDL) participate in atherosclerosis plaque formation, rupture, and subsequent thrombosis. Because oxLDL are toxic to cultured cells and Bcl-2 protein prevents apoptosis, the present work aimed to study whether Bcl-2 may counterbalance the toxicity of oxLDL. Two experimental model systems were used in which Bcl-2 levels were modulated: 1) lymphocytes in which the (high) basal level of Bcl-2 was reduced by antisense oligonucleotides; 2) HL60 and HL60/B (transduced by Bcl-2) expressing low and high Bcl-2 levels, respectively. In cells expressing relatively high Bcl-2 levels (lymphocytes and HL60/B), oxLDL induced mainly primary necrosis. In cells expressing low Bcl-2 levels (antisense-treated lymphocytes, HL60 and ECV-304 endothelial cells), the rate of oxLDL-induced apoptosis was higher than that of primary necrosis. OxLDL evoked a sustained calcium rise, which is a common trigger to necrosis and apoptosis since both types of cell death were blocked by the calcium chelator EGTA. Conversely, a sustained calcium influx elicited by the calcium ionophore A23187 induced necrosis in cells expressing high Bcl-2 levels and apoptosis in cells expressing low Bcl-2 levels. This suggests that Bcl-2 acts downstream from the calcium peak and inhibits only the apoptotic pathway, not the necrosis pathway, thus explaining the apparent shift from oxLDL-induced apoptosis toward necrosis when Bcl-2 is overexpressed.

Apoptosis and activation of the sphingomyelin-ceramide pathway induced by oxidized low density lipoproteins are not causally related in ECV-304 endothelial cells.

Oxidized low density lipoproteins (oxLDL) are thought to play a central role in the development of atherosclerosis. Toxic concentrations of mildly oxidized LDL elicit massive apoptosis of endothelial cells (Escargueil-Blanc, I., Meilhac, O., Pieraggi, M. T. , Arnal J. F., Salvayre, R., Nègre-Salvayre, A. (1997) Arterioscler. Thromb. Vasc. Biol. 17, 331-339). Since the lipid mediator ceramide emerged as a potent inducer of apoptosis, we aimed at investigating the occurrence of ceramide formation and its potential role in oxLDL-induced apoptosis. In ECV-304 endothelial cells, toxic concentrations of oxLDL triggered an early activation of the sphingomyelin-ceramide pathway, as shown by both sphingomyelin hydrolysis and ceramide formation. N-Tosyl-L-phenylalanyl chloromethyl ketone (TPCK) and dichloroisocoumarin (DCIC), two serine-protease inhibitors (serpins), blocked the oxLDL-induced ceramide generation but, unexpectedly, did not inhibit the oxLDL-induced apoptosis. Conversely, treatment of endothelial cells by bacterial sphingomyelinase, under conditions effectively generating ceramide, did not induce apoptosis. In contrast, short-chain permeant C2- and C6-ceramides elicited apoptosis of ECV-304. However, the mechanisms of apoptosis triggered by C2-ceramide and by oxLDL were (at least in part) different, because C2-ceramide-induced apoptosis was calcium-independent, whereas oxLDL-induced apoptosis was calcium-dependent. In conclusion, it is suggested that oxLDL-induced apoptosis is calcium-dependent but independent of the activation of the sphingomyelin-ceramide pathway and that the toxic effect of short chain permeant ceramides is calcium-independent and does not mimic the effect of natural ceramides induced by oxLDL.

Potential role for ceramide in mitogen-activated protein kinase activation and proliferation of vascular smooth muscle cells induced by oxidized low density lipoprotein.

Proliferation of vascular smooth muscle cells (SMC) is a hallmark in the pathogenesis of atherosclerotic lesions. Mildly oxidized low density lipoproteins (UV-oxLDL), which are mitogenic to cultured AG-08133A SMC, activate the sphingomyelin (SM)-ceramide pathway. We report here the following. (i) UV-oxLDL elicited a biphasic and sustained activation of MBP kinase activity, phosphorylation and nuclear translocation of p44/42 mitogen-activated protein kinase (MAPK), and [3H]thymidine incorporation, which were inhibited by PD-098059, a MAPK kinase inhibitor. (ii) The use of preconditioned media (from SMC pre-activated by UV-oxLDL) transferred to native SMC and blocking antibodies against growth factors suggest that UV-oxLDL-induced activation of MAPK and [3H]thymidine incorporation seem to be independent of any autocrine secretion of growth factors. (iii) UV-oxLDL-induced activation of a neutral sphingomyelinase, SM hydrolysis, ceramide production, and [3H]thymidine incorporation were inhibited by two serine-protease inhibitors (serpins), suggesting that a serpin-sensitive proteolytic pathway is involved in the activation of the SM-ceramide signaling pathway. (iv) UV-oxLDL-induced MAPK activation and [3H]thymidine incorporation were mimicked by ceramide generated in the plasma membrane by bacterial sphingomyelinase treatment or by addition of the permeant C2-ceramide. Serpins did not inhibit the MAPK activation and [3H]thymidine incorporation induced by C2-ceramide, indicating that activation of the MAPK and [3H]thymidine incorporation is subsequent to the stimulation of the SM-ceramide pathway. Taken together, these data suggest that mitogenic concentrations of UV-oxLDL are able to stimulate the SM-ceramide pathway through a protease-dependent mechanism and activate p44/42 MAPK, leading to proliferation of vascular SMC.

Effect of dietary phenolic compounds on apoptosis of human cultured endothelial cells induced by oxidized LDL.

1. Oxidized low density lipoproteins (LDL) are toxic to cultured endothelial cells. Mildly oxidized LDL, characterized by relatively low levels of TBARS and only minor modifications of apoB, were obtained by using 2 experimental model systems of oxidation, namely oxidation by u.v. radiation or ferrylmyoglobin (a two electron oxidation product from the reaction of metmyoglobin with H2O2). 2. Toxic concentrations of mildly oxidized LDL induce apoptosis (programmed cell death) of cultured endothelial cells, as shown by typical morphological features, by the in situ TUNEL procedure and by DNA fragmentation revealed on gel electrophoresis. This apoptosis is calcium-dependent and subsequent to the intense and sustained cytosolic [Ca2+]i peak elicited by oxidized LDL. 3. Five naturally occurring phenolic compounds present in food and beverages were able to prevent, in a concentration-dependent manner, the apoptosis of endothelial cells induced by oxidized LDL. Among the compounds tested, caffeic acid was the most effective. Under the conditions used, the protective effect of caffeic acid (IC50 8.3+/-2.1 micromol l[-1]) in the prevention of apoptosis induced by oxidized LDL was significantly higher than that of the other compounds tested (IC50s were 12.4+/-3.2, 14.1+/-4.1, 20.4+/-4.4 and 72.6+/-9.2 micromol l(-1) for ferulic, protocatechuic, ellagic and p-coumaric acids, respectively). 4. The anti-apoptotic effect of caffeic acid results from the addition of two effects, (i) the antioxidant effect which prevents LDL oxidation and subsequent toxicity ('indirect' protective effect); (ii) a 'direct' cytoprotective effect, acting at the cellular level. 5. Effective concentrations of caffeic acid acted at the cellular level by blocking the intense and sustained cytosolic [Ca2+]i rise elicited by oxidized LDL. 6. In conclusion, phenolic acids (caffeic and ferulic acids being the most potent of the compounds tested under the conditions used) exhibit a potent cytoprotective effect of cultured endothelial cells against oxidized LDL. In addition to antioxidant effect delaying LDL oxidation, caffeic acid acts as a cytoprotective agent, probably by blocking the intracellular signalling triggered by oxidized LDL and culminating in the sustained calcium rise which is involved in oxidized LDL-induced apoptosis.

HDL and ApoA prevent cell death of endothelial cells induced by oxidized LDL.

We have previously demonstrated that toxic doses of mildly oxidized LDL evokes in cultured cells a delayed and sustained rise of cytosolic [Ca2+], eliciting in turn irreversible cell damage and leading finally to cell death. HDL and delipidated apolipoprotein (apo). A prevented effectively the toxic effect of oxidized LDL to bovine aortic endothelial cells, in a time- and dose-dependent manner. The major part of the protective effect was mimicked by purified apoA-I, whereas purified apoA-II exhibited only very low protective activity. The protective effect was independent of the paraoxonase-linked HDL activity. The protective effect of HDL is independent of the contact of HDL with oxidized LDL, as shown by preincubation of oxidized LDL with HDL or apoA. In contrast, the protective effect was dependent on the integrity of apoA and on the contact of HDL with cells, thus suggesting that HDL acts directly on cells by enhancing their resistance against oxidized LDL. Preincubation experiments show that the protective effect is dependent on the duration of the contact of cells with HDL (maximal effect observed after 12 to 16 hours' preincubation), is also dependent on protein synthesis, and is persistent for at least 48 hours after the end of the contact of HDL with cells. Finally, effective concentrations of HDL inhibit the Ca2+ peak, which is directly involved in the cytotoxic effect of oxidized LDL, as shown by the inhibitory effect of Ca2+ chelators. All together, these results suggest that HDL, mainly apoA-I, increases the resistance of endothelial cells against oxidized LDL and prevents its toxic (apoptotic) effect by blocking the pathogenic intracellular signaling (culminating in sustained Ca2+ rise) involved in cell death.

Mitochondrial function is involved in LDL oxidation mediated by human cultured endothelial cells.

Human endothelial cells (ECs) grown under standard conditions are able to generate a basal level of oxygen free radicals and induce progressive oxidation of LDLs. Inhibition of cell-mediated LDL oxidation by superoxide dismutase, EDTA, or desferrioxamine implicates a role for superoxide anion and/or transition metals in this process. The potential role of the mitochondrion was investigated by inducing mitochondrial deenergization by selective photosensitization or the addition of inhibitors of the mitochondrial respiratory chain. Mitochondria of human cultured ECs were selectively damaged by photosensitization of cells labeled with the mitochondrion-selective fluorescent dye 2-(4-dimethylaminostyryl)-1-methylpyridinium iodide under conditions that induced only low levels of toxicity during the time of the experiment. Photosensitized ECs exhibited severe mitochondrial dysfunction, as suggested by the defect in mitochondrial uptake of the mitochondrion-selective fluorescent dyes [rhodamine 123 and 2-(4-dimethylaminostyryl)-1-methylpyridinium iodide] and morphological alterations as shown by transmission electron microscopy. In mitochondria-photosensitized cells, superoxide anion generation was strongly decreased, as was LDL oxidation and the subsequent cytotoxicity. When ECs were incubated with the mitochondrial respiratory-chain inhibitors antimycin A or rotenone or with the carbonylcyanide-m-chlorophenylhydrazone uncoupler rhodamine 123, uptake and subcellular distribution were altered, and concomitantly superoxide anion production and LDL oxidation were strongly decreased. In conclusion, these data suggest that mitochondrial function is required, directly or indirectly, for the production of superoxide anion and the subsequent LDL oxidation by human vascular ECs.

Oxidized LDLs induce massive apoptosis of cultured human endothelial cells through a calcium-dependent pathway. Prevention by aurintricarboxylic acid.

Oxidized LDLs are thought to play a central role in atherogenesis. Among their wide variety of biological properties, oxidized LDLs exhibit a cytotoxic effect on cultured vascular cells. Toxic doses of mildly oxidized LDLs elicited massive apoptosis in both primary and immortalized cultures of endothelial cells as shown by characteristic morphological and biochemical changes. Cytoplasmic and nucleic modifications (eg, chromatin condensation and nucleus fragmentation) were visualized by using electron and fluorescence microscopy of intact cells labeled by the fluorescent DNA probe SYTO-11. DNA fragmentation was quantified by ultracentrifugation of chromatin fragments, evaluated in situ by using the TUNEL (Terminal transferase-mediated dUTP-biotin nick end labeling) procedure, and visualized by electrophoresis of radiolabeled DNA fragments showing the characteristic apoptotic ladder. Apoptotic cells became rapidly detached and underwent postapoptotic necrosis that led to cell disintegration. Apoptosis was subsequent to a sustained and delayed peak of cytosolic calcium. Both the calcium peak and apoptosis were blocked by chelating the extracellular calcium with EGTA or by inhibiting the calcium influx by the calcium-channel blockers nifedipine and nisoldipine, thus suggesting that the apoptotic process induced by oxidized LDLs is clearly calcium dependent. Aurintricarboxylic acid, an inhibitor of endonucleases, also blocked the apoptotic process without blocking the calcium peak. These results suggest that toxic doses of mildly oxidized LDLs induce massive apoptosis of endothelial cells through a calcium-dependent mechanism and that this apoptotic process can be prevented by inhibiting the rise of cytosolic calcium or by inhibiting cellular endonucleases by aurintricarboxylic acid.

Necrosis and apoptosis induced by oxidized low density lipoproteins occur through two calcium-dependent pathways in lymphoblastoid cells.

Oxidized low density lipoproteins (LDL) elicit in cultured lymphoblastoid cell lines a delayed and sustained calcium rise followed by a progressive of DNA fragmentation, endogenous proteolysis, and morphological features of necrosis and apoptosis. All these events were blocked by chelating the calcium of the culture medium by EGTA, thus suggesting that the two types of cell death induced by oxidized LDL (necrosis and apoptosis) were subsequent to the rise in calcium. The protease inhibitors leupeptin and antipain were able to block (at least in part) the endogenous proteolysis and the necrotic process, but exhibited no effect on apoptosis and DNA fragmentation. At the opposite, aurintricarboxylic acid and spermine (inhibitors of DNA degradation by endonucleases) inhibit DNA fragmentation and morphological apoptosis, but not endogenous proteolysis and necrosis. These data suggest that cell death induced by oxidized LDL occurs through two calcium-dependent processes triggering 1) on one hand, proteolysis and subsequently necrosis characterized by the loss of cell membrane integrity; and 2) on the other hand, internucleosomal DNA cleavage and subsequently morphological apoptosis. The RNA or protein synthesis inhibitors, actinomycin D and cycloheximide, were completely ineffective in preventing endogenous proteolysis, DNA fragmentation, necrosis, and apoptosis induced by oxidized LDL. The subclassification of the type of apoptosis elicited by oxidized LDL is discussed.