Role of Phagocytosis in the Pro-Inflammatory Response in LDL-Induced Foam Cell Formation; a Transcriptome Analysis

Abstract

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.

Overview of OxLDL and Its Impact on Cardiovascular Health: Focus on Atherosclerosis

“…Modified LDL particles tend to self-associate, and these self-associates have the size quite analogues to the bacterial and other pathogens. Due to this, the phagocytosis of resident subendothelial cells of the arterial wall can be stimulated ( Orekhov et al, 2020a ).…”

Signaling Pathways and Key Genes Involved in Regulation of foam Cell Formation in Atherosclerosis

“…Several recent studies investigated the role of mollified LDL [53,54]. Native LDL, multiple modified LDL isolated from the blood of atherosclerotic patients, and LDL modified in vitro by desialylation, oxidation, and acetylation were used.…”

“…A recent study revealed the genes encoding inflammatory molecules that were up-regulated upon treatment of macrophages with atherogenic modified LDL and were involved in the development of inflammation [40]. Further knock-down experiments elucidated the relationship between the immune response and cholesterol accumulation [53]. Primary human macrophages were cultured with atherogenic naturally occurring multiply-modified LDL that induced a prominent cholesterol accumulation.…”

Lipid Metabolism in Macrophages: Focus on Atherosclerosis

“…Aggregated modified LDL (agLDL) may cause lipid accumulation in macrophages in two ways. First, it is known that agLDL is recognized by LRP1 and engulfed via phagocytosis [ 12 , 13 ]. Second, as it was shown recently, macrophages may provide degradation of agLDL via lysosomal synapses, which are extracellular hydrolytic compartments [ 14 ].…”