Study On Mechanism Associated With The Kinetics Of Formation And Damage In Early Macrophage Foam Cell Induced By Oxidized Low-density Lipoprotein

Atherosclerosis is a multifactorial disease that is associated with many risk factors including dyslipidemia, diabetes, obesity and hypertension. The accumulation of Oxidized low density lipoprotein (Ox-LDL) in the arterial wall constitutes a fundamental event in atherogenesis. Macrophages play unique and complex roles in arterial wall cholesterol metabolism. At early stages of lipid infiltration, macrophages take up Ox-LDL via the scavenger receptor (CD36) pathway, resulting in foam cell formation. Meanwhile, Ox-LDL increased mediators of inflammation, altered cell morphology and the transduction of signals, and damaged cells. Therefore, the formation and damage of macrophage foam cells is a characteristic feature of early atherosclerotic lesions, spatiotemporal kinetic mechanisms by which Ox-LDL may trigger have not been completely understood.To address this question, the human monocyte U937-derived macrophages by Phorbol 12-myristate 13-acetate (PMA) were chosen as target cells in the current study. By the use of confocal laser microscope, flow cytometer, molecular biological methods and based on kinetic data analysis, under the acute and chronic exposure of U937-derived macrophages to Ox-LDL, the kinetic mechanism about the formation and damage of early macrophage foam cells was studied.In the spatiotemporal dynamic view, we have detected cholesteryl ester (CE), total cholesterol (TC), the assembly of F-actin and the expression of CD36 and CD54, intracellular free Ca~2+ ([Ca~2+]i) associated with the formation of macrophage foam cells. The cholesteryl ester and free cholesterol increased significantly by Ox-LDL in time-dependent manner. It is noteworthy that the ratio of (cholesteryl ester)/(total cholesterol) at 24h exceeded 50%, applying that U937-derived macrophages were transformed into typical foam cells at 24 h treatments. The uptake of Ox-LDL significantly increased [Ca~2+]i in U937-derived macrophages in both acute and chronic treatments (P<0.01). In particular, the increases of the induced [Ca~2+]i were different in the presence or absence of extracellular Ca~2+ under the acute exposure.There was a transmembrane Ca~2+ gradient across the nuclear membrane in macrophages following their transformation to foam cells. A time-dependent rise in F-actin assembly and expression of CD36 and CD54 at 12h and 24h was induced respectively by Ox-LDL.Otherwise, from the spatiotemporal dynamics, we have studied a complex interaction of mitochondrial respiration, cell apoptosis and necrosis, mitochondrial membrane potential (A\|/m), glutathione (GSH), the expression of BcI-2 and imbalance of intracellular peroxides (H2O2), nitric oxide (NO) and superoxide (O2") associated with the damage of macrophage foam cells. The uptake of Ox-LDL significantly increased production of intracellular NO and O2" in U937-derived macrophages in both acute and chronic treatments. Based on kinetic data analysis, the fluorescence intensity changes of NO were more significant than of O2" in acute exposure of cells to Ox-LDL, but after 2min the increase of O2" became slow. Moreover, the increase of intracellular H2O2 was observed in acute treatments. But at 24 h, the pronounced decline of intracellular H2O2 was found. Ox-LDL induced cell apoptosis and necrosis, reduced mitochondrial respiration, stimulated mitochondrial membrane hyperpolarization, and caused adaptive increase of glutathione synthesis in time-dependent manner. But at 24 h, Ox-LDL partially resulted in mitochondrial membrane depolarization, and caused the decrease of ROS and GSH. In contrast with control cells, the expression of Bcl-2 at 12h and 24h was increased by Ox-LDL. But at 24h, the expression of Bcl-2 significantly decreased than at 12h (From 500-600% to 300-400%).Based on above results, it is reasonable to draw conclusion as below: 1) In the formation of early macrophage foam cells, the spatiotemporal increases of [Ca2+]i induced quickly by Ox-LDL could one of more early cell events. Meanwhile, in time-dependent manner, Ox-LDL increased the expression of CD36 and CD54, induced the spatiotemporal rearrangement of cytoskeleton, affected cellular phagocytosis and adhesion, and resulted in the formation of typical foam cells at 24 h; 2) In the damage of early macrophage foam cells, A time-dependenct rise of NO and O2" was induced by Ox-LDL. Moreover, Ox-LDL increased the release of H2O2,caused adaptive increase of glutathione (GSH) synthesis and expression of Bcl-2, stimulated mitochondrial membrane hyperpolarization, reduced mitochondrial respiration, and induced cell apoptosis and necrosis at 12 h, applying that antioxidant defense systems in nucleus involved in cell damage; 3) Otherwise, with further damage, Ox-LDL caused the decrease of glutathione and H2O2, expression of Bcl-2 and partial mitochondrial membrane depolarization. Ox-LDL reduced mitochondrial respiration, and induced cell apoptosis and necrosis with time. The results suggested that mitochondrial dysfunction might play a critical function in the early atherosclerotic lesions. Therefore, in the spatiotemporal kinetic view, the study elucidated that the formation and damage of macrophage foam cells are a kinetic process which has target factors and networks. The findings suggest that modulation of target factors metabolism may be a potential therapeutic strategy against atherosclerosis.