benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone and Atherosclerosis

In the immune response, hypohalous acids are generated by activated leukocytes via the release of myeloperoxidase and the formation of H2O2. Although these oxidants have important bactericidal properties, they have also been implicated in causing tissue damage in inflammatory diseases, including atherosclerosis. Hypochlorous acid (HOCl) and hypothiocyanous acid (HOSCN) are the major oxidants formed by myeloperoxidase under physiological conditions, with the ratio of these oxidants dependent on diet and smoking status. HOCl is highly reactive and causes marked cellular damage, but few data are available on the effects of HOSCN on mammalian cells. In this study, we have compared the actions of HOCl and HOSCN on human coronary artery endothelial cells (HCAEC). HOCl reacts rapidly with the cells, resulting in extensive cell death by both apoptosis and necrosis, with necrosis dominating at higher oxidant doses. In contrast, HOSCN is consumed more slowly, with cell death occurring only by apoptosis. Exposure of HCAEC to HOCl and HOSCN induces changes in mitochondrial membrane permeability, which, in the case of HOSCN, is associated with mitochondrial release of proapoptotic factors, including cytochrome c, apoptosis-inducing factor, and endonuclease G. With each oxidant, apoptosis appears to be caspase-independent, with the inactivation of caspases 3/7 observed, and pretreatment of the cells with the caspase inhibitor Z-VAD-fmk having no effect on the extent of cell death. Loss of cellular thiols, depletion of glutathione, and the inactivation of thiol-dependent enzymes, including glyceraldehyde-3-phosphate dehydrogenase, were seen with both oxidants, though to a much greater extent with HOCl. The ability of myeloperoxidase-derived oxidants to induce endothelial cell apoptosis may contribute to the formation of unstable lesions in atherosclerosis. The results with HOSCN may be particularly significant for smokers, who have elevated plasma levels of SCN(-), the precursor of this oxidant.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Apoptosis Inducing Factor; Atherosclerosis; Caspase 3; Caspase 7; Caspase Inhibitors; Cell Line; Cell Survival; Coronary Vessels; Cytochromes c; Endodeoxyribonucleases; Endothelial Cells; Glutathione; Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating); Humans; Hydrogen Peroxide; Hypochlorous Acid; Mitochondrial Membranes; Necrosis; Oxidation-Reduction; Permeability; Peroxidase; Sulfhydryl Compounds; Thiocyanates

Chronic kidney disease with hyperphosphatemia is associated with accelerated atherosclerosis and endothelial dysfunction. However, the contribution of high serum phosphate levels to endothelial injury is incompletely understood. The aim of this work was to evaluate the responses of endothelial cells to elevated levels of extracellular phosphate in vitro. High phosphate in concentrations similar to those observed in uremia-associated hyperphosphatemia (>2.5 mM) induced apoptosis in two endothelial cell lines (EAhy926 cells and GM-7373 cells). This effect was enhanced when cells were incubated for 24 h in the presence of 2.8 mM calcium instead of 1.8 mM. By treating cells with 0.5 or 1.0 mM phosphonoformic acid, an inhibitor of the phosphate transporter, death was completely prevented. The process of phosphate-induced apoptosis was further characterized by increased oxidative stress, as detected by increased ROS generation and disruption of the mitochondrial membrane potential at approximately 2 h after treatment, followed by caspase activation. These findings show that hyperphosphatemia causes endothelial cell apoptosis, a process that impairs endothelial integrity. Endothelial cell injury induced by high phosphate concentrations may be an initial event leading to vascular complications in patients with chronic kidney disease.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Atherosclerosis; Calcium; Caspase Inhibitors; Cells, Cultured; Cysteine Proteinase Inhibitors; Endothelial Cells; Gene Expression; Humans; Hyperphosphatemia; In Vitro Techniques; Kidney Failure, Chronic; Membrane Potential, Mitochondrial; Phosphates; Reactive Oxygen Species; Sodium-Phosphate Cotransporter Proteins; Umbilical Veins

Several lines of evidence suggest that macrophages play a key role in atherosclerotic plaque destabilization and rupture. Therefore, selective removal of macrophages from plaques via pharmacological therapy could represent a promising approach to stabilize "vulnerable," rupture-prone lesions. Yet, how macrophages can be eliminated from plaques without influencing other cell types, including smooth muscle cells (SMCs), is unknown. In the present study, we report that benzyloxycarbonyl-Val-Ala-DL-Asp(O-methyl)-fluoromethylketone (z-VAD-fmk), a caspase inhibitor with broad specificity, induces nonapoptotic cell death of J774A.1 and RAW264.7 macrophages but not of SMCs. Cell death was characterized by bulk degradation of long-lived proteins, processing of microtubule-associated protein light chain 3, and cytoplasmic vacuolization, which are all markers of autophagy. However, necrosis also occurred, and the number of necrotic cells rapidly increased during z-VAD-fmk treatment. Primary mouse peritoneal macrophages were resistant to z-VAD-fmk-mediated cell death, but unlike SMCs, they underwent z-VAD-fmk-mediated necrosis after pretreatment with interferon-gamma. Further evidence indicated that the expression level of receptor-interacting protein 1 (RIP1) mediates the sensitivity to z-VAD-fmk. Importantly, upon z-VAD-fmk treatment, J774A.1 macrophages overexpressed and secreted several chemokines and cytokines, including tumor necrosis factor (TNF) alpha. The combination of z-VAD-fmk and TNFalpha, but not TNFalpha alone, induced SMCs necrosis via a mechanism that required RIP1 expression. These results suggest that z-VAD-fmk, despite its selective cell death inducing capacity, would be detrimental for the stability of atherosclerotic plaques due to enlargement of the necrotic core, stimulation of inflammatory responses, and indirect induction of SMC death.

Topics: Amino Acid Chloromethyl Ketones; Animals; Atherosclerosis; Cell Line; Cytokines; Macrophages, Peritoneal; Mice; Mice, Inbred C57BL; Microscopy, Electron; Muscle, Smooth, Vascular; Necrosis; Protein Serine-Threonine Kinases; Receptor-Interacting Protein Serine-Threonine Kinases; Reverse Transcriptase Polymerase Chain Reaction; Tumor Necrosis Factor Receptor-Associated Peptides and Proteins