According to the state of mitochondrial respiration, the respiratory chain generates superoxide anions converted into hydrogen peroxide. Two uncoupling proteins (UCP) able to modulate the coupling between the respiratory chain and ATP synthesis are now identified and could be involved in mitochondrial H2O2 generation. UCP1 is specific to brown adipose tissue (BAT) whereas UCP2 is expressed in numerous tissues, particularly in monocytes/macrophages. Preincubation of BAT mitochondrial fractions with GDP, an inhibitor of UCP1, induced a rise in mitochondrial membrane potential (assessed by rhodamine 123 uptake) and H2O2 production. An uncoupling agent reversed this effect. Liver mitochondria exhibited a similar phenotype. GDP was also able to raise membrane potential and H2O2 production of the mitochondria from nonparenchymal cells expressing UCP2, but was completely ineffective on mitochondria from hepatocytes deprived of UCP2. The GDP effect was also observed with mitochondrial fractions of the spleen or thymus, which highly expressed UCP2. Altogether, these results strongly suggest that UCP2 is sensitive to GDP and that the UCPs, particularly UCP2, are able to modulate H2O2 mitochondrial generation. This supports a role for UCP2 in cellular (patho-) physiological processes involving free radicals generated by mitochondria, such as oxidative damage, inflammation, or apoptosis.

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.

Abstract-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. Key Words: angiotensin II Ⅲ apoptosis Ⅲ calcium Ⅲ muscle, smooth, vascular Ⅲ cells V ascular smooth muscle cells (VSMCs) maintain vascular tone mainly through coordinated contraction/relaxation, and they play a role in arterial wall remodeling through proliferation, hypertrophy, and apoptosis. 1 The arterial tree exhibits heterogeneity in response to vasoactive stimuli and in alterations induced by hypertension or atherosclerosis. The concept that distinct VSMC subtypes may play specific roles, at different locations or pathophysiological situations, is well documented in animals, 2-6 and VSMC heterogeneity also exists in human arteries. 7 In the rat arterial model, 2 major VSMC subtypes have been discerned on the basis of stable distinct morphologies in culture: spindle versus epithelioid. 2,8,9 They also differ in growth properties or protein synthesis 2,8 -11 ; however, their responses to vasoactive stimuli are barely known. 12 Angiotensin (Ang) II, the main effector peptide of the renin-angiotensin system, plays an important role in normal vascular physiology and cardiovascular diseases, 13 mostly through the Ang II type 1 (AT 1 ) receptor and partly through the Ang II type 2 (AT 2 ) receptor. AT 1 receptor-mediated intracellular calcium concentration ([Ca 2ϩ ] i ) signaling is a major determinant of VSMC contraction. 14 Ang II is a modulator of VSMC growth with proliferative/hypertrophic effects mediated by the AT 1 receptor through complex, partly calcium-dependent, signaling. 14 Heterogeneity exists in Ang II calcium signaling 14 -16 and long-term responses 17-19 between individual VSMCs, arteries, or arterial layers. In vivo, Ang II can induce a delayed apoptosis through AT 1 receptor activation. 20,21 Inversely, in vitro, Ang II induces apoptosis through AT 2 receptor activation in AT 2 -transfected VSMCs, 22 whereas AT 1 receptors might also protect native VSMCs against acute NO-induced apoptosis. 23 The question of whether the diversity in Ang II biological effects could originate from variations in the different VSMC subtypes has not been addressed. Therefore, the aim of the present study was to investigate whether VSMC subtypes responded differently to Ang II in [Ca 2ϩ ] i signaling and long-term survival. We used 2 stable cell lines, Sp-SMC and Ep-SMC, as respective models of spindle and epithelioid rat arterial VSMC subtypes. The results show dramatically different Ang II responses in the 2 VSMC lines. A delayed AT 1 receptor-mediated calcium-dependent apoptosis was

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.

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