Objectives Cardiovascular disease is progressive inflammatory disease characterised by the formation of atherosclerotic plaques made up of lipid load macrophage cells within the artery walls. The death of these lipid loads cells by oxidised low density lipoprotein (oxLDL) causes plaque instability, leading to both plaque growth and plaque rupture. The cell death is caused by severe oxidative stress triggered by the oxLDL. The cell death is also associated with a rise in intracellular calcium. This study is examining the source of the calcium rise and whether it is a cause or consequence of the oxidative stress.
Methods Human monocytes prepared from whole human blood were used to study the role of calcium in oxLDL-mediated cytotoxicity. Cells were pre-incubated with fluorescent calcium binding probe- Fluo 3-AM before treated with oxLDL. Selected calcium channel blockers specific for plasma membrane, endoplasmic reticulum, mitochondria as well as calcium chelator were tested for their effects on the oxLDL mediated cell death process. The movement of calcium into the cytoplasm and changes in intracellular calcium was measured by flow cytometer using the Fluo 3-AM probe. Cell viability was determined by spectrophotometric measurement of MTT reduction and propidium iodide staining by flow cytometry. DHE staining was used to measure the formation of reactive oxygen species (ROS).
Results Human monocytestreated with oxLDL caused increasing intracellular free calcium. Cells incubated in calcium free media as well as media with calcium developed similar patterns of cell death. Blocking the calcium channels by incubating the cells with different types of inhibitors did not prevent cell death. However, inhibition of the calcium rise by a calcium chelator- EGTA, was able to impede intracellular calcium rise but not cell death. Higher DHE fluorescence levels detected in cells treated with oxLDL compared to the control cells showing the presence of ROS which are causing oxidative stress.
Conclusions With human monocyte cells, oxLDL-mediated cell death, is not dependent on an increase in intracellular calcium as blocking the calcium did not inhibit the cell death. However, an increase in intracellular calcium may therefore activate ROS that induced oxidative stress leading to cell death.