Redistribution of intracellular calcium and its effect on apoptosis in macrophages: Induction by oxidized LDL.
ABSTRACT Calcium signaling, as a key to early step of the elementary intracellular events, has been implicated in controlling the development of atherosclerosis. We have shown previously that oxidized low density lipoprotein OxLDL-induced spatiotemporal increases of intracellular free calcium ([Ca(2+)](i)) in the early formation of macrophage foam cells. Here, we evaluated how spatiotemporal redistribution of intracellular calcium occurs and would affect OxLDL-induced apoptosis. Confocal laser scanning microscopy and flow cytometry showed the time-dependent increase of mitochondrial Ca(2+) ([Ca(2+)](m)) in acute and chronic exposure of U937-derived macrophages to OxLDL (100 microg/ml). Independent of the presence or absence of external Ca(2+), OxLDL-induced a peak of [Ca(2+)](m) in acute exposure, whose amplitude in the absence of extracellular Ca(2+) was obviously lower than the presence of extracellular Ca(2+). In addition, the thapsigargin-mediated increase of [Ca(2+)](i), through endoplasmic reticulum (ER) Ca(2+) pump depletion, was obviously reduced by 1-h pretreatment of OxLDL. OxLDL also caused a time-dependent opening of mitochondrial permeability transition pores (PTPs). EGTA/AM, an intracellular Ca(2+) chelator, significantly reduced OxLDL-induced apoptosis and failed to prevent OxLDL-induced necrosis at 6h. In contrast to control cells, chelation of cytosolic Ca(2+) by EGTA/AM at 6h did not completely reverse OxLDL-induced apoptosis. OxLDL stimulated depolarization of mitochondrial membrane potential (Deltapsi) in time-dependent manner. Our data demonstrated that OxLDL-induced spatiotemporal Ca(2+) redistribution in appropriate organelles and mediated Ca(2+)-dependent apoptosis in relation to depolarization of Deltapsi. These findings suggested that manipulation of the intracellular calcium balance may be a useful strategy to limit the loss of macrophages in early atherosclerosis.
Article: Oxidized low-density lipoprotein-activated c-Jun NH2-terminal kinase regulates manganese superoxide dismutase ubiquitination: implication for mitochondrial redox status and apoptosis.[show abstract] [hide abstract]
ABSTRACT: Oxidized low-density lipoprotein (oxLDL) modulates intracellular redox status and induces apoptosis in endothelial cells. However, the signal pathways and molecular mechanism remain unknown. In this study, we investigated the role of manganese superoxide dismutase (Mn-SOD) on oxLDL-induced apoptosis via c-Jun NH2-terminal kinase (JNK)-mediated ubiquitin/proteasome pathway. OxLDL induced JNK phosphorylation that peaked at 30 minutes in human aortic endothelial cells. Fluorescence-activated cell sorting analysis revealed that oxLDL increased mitochondrial superoxide production by 1.88+/-0.19-fold and mitochondrial membrane potential by 18%. JNK small interference RNA (siJNK) reduced oxLDL-induced mitochondrial superoxide production by 88.4% and mitochondrial membrane potential by 61.7%. OxLDL did not affect Mn-SOD mRNA expression, but it significantly reduced Mn-SOD protein level, which was restored by siJNK. Immunoprecipitation by ubiquitin antibody revealed that oxLDL increased ubiquitination of Mn-SOD, which was inhibited by siJNK. OxLDL-induced caspase-3 activities were also attenuated by siJNK but were enhanced by Mn-SOD small interfering RNA. Furthermore, overexpression of Mn-SOD abrogated oxLDL-induced caspase-3 activities. OxLDL-induced JNK activation regulates mitochondrial redox status and Mn-SOD protein degradation via JNK-dependent ubiquitination, leading to endothelial cell apoptosis.Arteriosclerosis Thrombosis and Vascular Biology 02/2010; 30(3):436-41. · 6.37 Impact Factor
Article: Atherosclerosis: pathogenesis and increased occurrence in individuals with HIV and Mycobacterium tuberculosis infection[show abstract] [hide abstract]
ABSTRACT: Timothy Guilford1, Devin Morris2,4, Dennis Gray3,4, Vishwanath Venketaraman3,41Your Energy Systems, Palo Alto, CA, USA; 2Graduate of College of Biomedical Sciences, 3College of Osteopathic Medicine of the Pacific, 4Western University of Health Sciences, Pomona, CA, USAAbstract: Atherosclerosis is a leading cause of coronary heart disease and stroke. Since 1981, more than 980,000 cases of AIDS have been reported in the United States. According to the Centers for Disease Control, more than 1 million Americans may be infected with HIV. By killing or damaging CD4+ T cells of the body’s immune system, HIV progressively destroys the body’s ability to fight infections. People diagnosed with AIDS often suffer from life-threatening diseases caused by opportunistic infections such as tuberculosis. HIV-infected individuals have increased risks for atherosclerosis. This review summarizes the effects of oxidized low density lipoproteins in impairing macrophage functions in individuals with atherosclerosis (with and without HIV infection) thereby enhancing the susceptibility to Mycobacterium tuberculosis infection.Keywords: AIDS, HIV, Mycobacterium tuberculosisHIV/AIDS - Research and Palliative Care. 01/2010;
Article: Assessing mitochondrial redox status by flow cytometric methods: vascular response to fluid shear stress.[show abstract] [hide abstract]
ABSTRACT: Mitochondria are an important source of superoxide production contributing to physiological and pathological responses, including vascular oxidative stress that is relevant to cardiovascular diseases. Vascular oxidative stress is intimately linked with pro-inflammatory states and atherosclerosis. Oxidized low-density lipoprotein (OxLDL) modulates intracellular redox status and induces apoptosis in endothelial cells. Hemodynamic, specifically, fluid shear stress imparts both biomechanical and metabolic effects on vasculature. Mitochondria are an important source of superoxide production contributing to vascular oxidative stress with relevance to cardiovascular diseases. We hereby present biophysical and biochemical approaches, including fluorescence-activated cell sorting, to assess the dynamics of vascular redox status.Current protocols in cytometry / editorial board, J. Paul Robinson, managing editor ... [et al.] 10/2011; Chapter 9:Unit9.37.