Accumulation of lipid-laden (foam) cells in the arterial wall is known to be the earliest step in the pathogenesis of atherosclerosis. There is almost no doubt that atherogenic modified low-density lipoproteins (LDL) are the main sources of accumulating lipids in foam cells. Atherogenic modified LDL are taken up by arterial cells, such as macrophages, pericytes, and smooth muscle cells in an unregulated manner bypassing the LDL receptor. The present study was conducted to reveal possible common mechanisms in the interaction of macrophages with associates of modified LDL and non-lipid latex particles of a similar size. To determine regulatory pathways that are potentially responsible for cholesterol accumulation in human macrophages after the exposure to naturally occurring atherogenic or artificially modified LDL, we used transcriptome analysis. Previous studies of our group demonstrated that any type of LDL modification facilitates the self-association of lipoprotein particles. The size of such self-associates hinders their interaction with a specific LDL receptor. As a result, self-associates are taken up by nonspecific phagocytosis bypassing the LDL receptor. That is why we used latex beads as a stimulator of macrophage phagocytotic activity. We revealed at least 12 signaling pathways that were regulated by the interaction of macrophages with the multiple-modified atherogenic naturally occurring LDL and with latex beads in a similar manner. Therefore, modified LDL was shown to stimulate phagocytosis through the upregulation of certain genes. We have identified at least three genes (F2RL1, EIF2AK3, and IL15) encoding inflammatory molecules and associated with signaling pathways that were upregulated in response to the interaction of modified LDL with macrophages. Knockdown of two of these genes, EIF2AK3 and IL15, completely suppressed cholesterol accumulation in macrophages. Correspondingly, the upregulation of EIF2AK3 and IL15 promoted cholesterol accumulation. These data confirmed our hypothesis of the following chain of events in atherosclerosis: LDL particles undergo atherogenic modification; this is 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. This chain of events may explain the relationship between cholesterol accumulation and inflammation. The primary sequence of events in this chain is related to inflammatory response rather than cholesterol accumulation.

Recent studies have shown that cellular metabolism is tightly linked to the regulation of immune cells. Here, we show that activation of cholesterol metabolism, involving cholesterol uptake, synthesis, and autophagy/lipophagy, is integral to innate immune responses in macrophages. In particular, cholesterol accumulation within endosomes and lysosomes is a hallmark of the cellular cholesterol dynamics elicited by Toll-like receptor 4 activation and is required for amplification of myeloid differentiation primary response 88 (Myd88) signaling. Mechanistically, Myd88 binds cholesterol via its CLR recognition/interaction amino acid consensus domain, which promotes the protein’s self-oligomerization. Moreover, a novel supramolecular compound, polyrotaxane (PRX), inhibited Myd88‑dependent inflammatory macrophage activation by decreasing endolysosomal cholesterol via promotion of cholesterol trafficking and efflux. PRX activated liver X receptor, which led to upregulation of ATP binding cassette transporter A1, thereby promoting cholesterol efflux. PRX also inhibited atherogenesis in Ldlr ^–/– mice. In humans, cholesterol levels in circulating monocytes correlated positively with the severity of atherosclerosis. These findings demonstrate that dynamic changes in cholesterol metabolism are mechanistically linked to Myd88‑dependent inflammatory programs in macrophages and support the notion that cellular cholesterol metabolism is integral to innate activation of macrophages and is a potential therapeutic and diagnostic target for inflammatory diseases.

Mitochondrial diseases (MD) are a heterogeneous group of multisystem disorders involving metabolic errors. MD are characterized by extremely heterogeneous symptoms, ranging from organ-specific to multisystem dysfunction with different clinical courses. Most primary MD are autosomal recessive but maternal inheritance (from mtDNA), autosomal dominant, and X-linked inheritance is also known. Mitochondria are unique energy-generating cellular organelles designed to survive and contain their own unique genetic coding material, a circular mtDNA fragment of approximately 16,000 base pairs. The mitochondrial genetic system incorporates closely interacting bi-genomic factors encoded by the nuclear and mitochondrial genomes. Understanding the dynamics of mitochondrial genetics supporting mitochondrial biogenesis is especially important for the development of strategies for the treatment of rare and difficult-to-diagnose diseases. Gene therapy is one of the methods for correcting mitochondrial disorders.Graphical 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.

The Mediator complex is a multi-subunit protein complex which plays a significant role in the regulation of eukaryotic gene transcription. It provides a platform for the interaction of transcriptional factors and RNA polymerase II, thus coupling external and internal stimuli with transcriptional programs. Molecular mechanisms underlying Mediator functioning are intensively studied, although most often using simple models such as tumor cell lines and yeast. Transgenic mouse models are required to study the role of Mediator components in physiological processes, disease, and development. As constitutive knockouts of most of the Mediator protein coding genes are embryonically lethal, conditional knockouts and corresponding activator strains are needed for these studies. Recently, they have become more easily available with the development of modern genetic engineering techniques. Here, we review existing mouse models for studying the Mediator, and data obtained in corresponding experiments.