Excessive accumulation of lipid inclusions in the arterial wall cells (foam cell 
formation) caused by modified low-density lipoprotein (LDL) is the earliest and 
most noticeable manifestation of atherosclerosis. The mechanisms of foam cell 
formation are not fully understood and can involve altered lipid uptake, 
impaired lipid metabolism, or both. Recently, we have identified the top 10 
master regulators that were involved in the accumulation of cholesterol in 
cultured macrophages induced by the incubation with modified LDL. It was found 
that most of the identified master regulators were related to the regulation of 
the inflammatory immune response, but not to lipid metabolism. A possible 
explanation for this unexpected result is a stimulation of the phagocytic 
activity of macrophages by modified LDL particle associates that have a 
relatively large size. In the current study, we investigated gene regulation in 
macrophages using transcriptome analysis to test the hypothesis that the primary 
event occurring upon the interaction of modified LDL and macrophages is the 
stimulation of phagocytosis, which subsequently triggers the pro-inflammatory 
immune response. We identified genes that were up- or downregulated following 
the exposure of cultured cells to modified LDL or latex beads (inert 
phagocytosis stimulators). Most of the identified master regulators were 
involved in the innate immune response, and some of them were encoding major 
pro-inflammatory proteins. The obtained results indicated that pro-inflammatory 
response to phagocytosis stimulation precedes the accumulation of intracellular 
lipids and possibly contributes to the formation of foam cells. In this way, the 
currently recognized hypothesis that the accumulation of lipids triggers the 
pro-inflammatory response was not confirmed. Comparative analysis of master 
regulators revealed similarities in the genetic regulation of the interaction of 
macrophages with naturally occurring LDL and desialylated LDL. Oxidized and 
desialylated LDL affected a different spectrum of genes than naturally occurring 
LDL. These observations suggest that desialylation is the most important 
modification of LDL occurring in vivo. Thus, modified LDL caused the gene 
regulation characteristic of the stimulation of phagocytosis. Additionally, the 
knock-down effect of five master regulators, such as IL15, EIF2AK3, F2RL1, 
TSPYL2, and ANXA1, on intracellular lipid accumulation was tested. We knocked 
down these genes in primary macrophages derived from human monocytes. The 
addition of atherogenic naturally occurring LDL caused a significant 
accumulation of cholesterol in the control cells. The knock-down of the EIF2AK3 
and IL15 genes completely prevented cholesterol accumulation in cultured 
macrophages. The knock-down of the ANXA1 gene caused a further decrease in 
cholesterol content in cultured macrophages. At the same time, knock-down of 
F2RL1 and TSPYL2 did not cause an effect. The results obtained allowed us to 
explain in which way the inflammatory response and the accumulation of 
cholesterol are related confirming our hypothesis of atherogenesis development 
based on the following viewpoints: LDL particles undergo atherogenic 
modifications that, in turn, accompanied by the formation of self-associates; 
large LDL associates stimulate phagocytosis; as a result of phagocytosis 
stimulation, pro-inflammatory molecules are secreted; these molecules cause or 
at least contribute to the accumulation of intracellular cholesterol. Therefore, 
it became obvious that the primary event in this sequence is not the 
accumulation of cholesterol but an inflammatory response.