cytochalasin-d and Atherosclerosis

It has been determined through extensive studies that autophagy, the Nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome and apoptotic responses in macrophages jointly contribute to atherogenesis and its development in the presence of lipid abnormalities. Few studies have investigated in full-scale if the intervention time for lipids abnormality or NLRP3 activation have a significant effect on autophagy, NLRP3 or the apoptotic status in macrophages.. Human THP-1 monocyte-derived macrophages were established by challenging THP-1 monocytes with 80 µg/ml oxidized low-density lipoprotein (ox-LDL) for specific durations. Foam cell formation was observed by Oil Red O (ORO) staining. Western blots were employed to determine protein expression. Transmission electron microscope (TEM) and immunofluorescence microscopy were applied to observe the autophagic status of cells. Cell apoptosis was evaluated by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL).. The cells were treated with ox-LDL for 12 h and 36 h, which were considered to represent early and advanced stages of atherogenesis for this study. The results showed that inhibition of ox-LDL phagocytosis by cytochalasin D in the early stage improved autophagic status, reduced NLRP3 activation and the apoptotic response significantly. In contrast, cytochalasin D had little effect on blocking the detrimental effect of ox-LDL at the advanced stage. Moreover, the changes in autophagy, apoptosis and NLRP3 expression after treatment with small interfering (si) RNA targeting NLRP3 in the early and advanced stages of atherogenesis were consistent with the above data.. Interventions against lipid disorders or inflammatory reactions in the early or advanced stages of atherogenesis may have different results depending on when they are applied during the process of atherosclerotic pathogenesis. These results may help improve therapeutic strategies for atherosclerosis prevention. Furthermore, a healthy lifestyle should still be recommended as the most important and inexpensive measure to prevent atherogenesis.

Topics: Apoptosis; Atherosclerosis; Autophagy; Cytochalasin D; DNA Nucleotidylexotransferase; Humans; Inflammasomes; Lipoproteins, LDL; Macrophages; NLR Family, Pyrin Domain-Containing 3 Protein; Nucleotides; RNA

A key event during the formation of lipid-rich foam cells during the progression of atherosclerosis is the uptake of modified low-density lipoproteins (LDL) by macrophages in response to atherogenic mediators in the arterial intima. In addition to scavenger receptor-dependent uptake of LDL, macropinocytosis is known to facilitate the uptake of LDL through the constitutive and passive internalization of large quantities of extracellular solute. In this study we confirm the ability of macropinocytosis to facilitate the uptake of modified LDL by human macrophages and show its modulation by TGF-β, IFN-γ, IL-17A and IL-33. Furthermore we show that the TGF-β-mediated inhibition of macropinocytosis is a Smad-2/-3-independent process.

Topics: Atherosclerosis; Biological Transport; Cell Differentiation; Cells, Cultured; Cytochalasin D; Foam Cells; Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating); Humans; Inflammation; Interferon-gamma; Interleukin-17; Interleukin-33; Interleukins; Lipoproteins, LDL; Macrophages; Nucleic Acid Synthesis Inhibitors; Pinocytosis; RNA Interference; RNA, Small Interfering; Smad2 Protein; Smad3 Protein; Transforming Growth Factor beta

Oral viridans group streptococci are the major commensal bacteria of the supragingival oral biofilm and have been detected in human atheromatous plaque. Atherosclerosis involves an ongoing inflammatory response, reportedly involving chronic infection caused by multiple pathogens. The aim of this study was to examine the invasion of human aortic endothelial cells (HAECs) by oral viridans group streptococci and the subsequent cytokine production by viable invaded HAECs. The invasion of HAECs by bacteria was examined using antibiotic protection assays and was visualized by confocal scanning laser microscopy. The inhibitory effects of catalase and cytochalasin D on the invasion of HAECs were also examined. The production of cytokines by invaded or infected HAECs was determined using enzyme-linked immunosorbent assays, and a real-time polymerase chain reaction method was used to evaluate the expression of cytokine messenger RNA. The oral streptococci tested were capable of invading HAECs. The number of invasive bacteria increased with the length of the co-culture period. After a certain co-culture period, some organisms were cytotoxic to the HAECs. Catalase and cytochalasin D inhibited the invasion of HAECs by the organism. HAECs invaded by Streptococcus mutans Xc, Streptococcus gordonii DL1 (Challis), Streptococcus gordonii ATCC 10558 and Streptococcus salivarius ATCC 13419 produced more cytokine(s) (interleukin-6, interleukin-8, monocyte chemoattractant protein-1) than non-invaded HAECs. The HAECs invaded by S. mutans Xc produced the largest amounts of cytokines, and the messenger RNA expression of cytokines by invaded HAECs increased markedly compared with that by non-invaded HAECs. These results suggest that oral streptococci may participate in the pathogenesis of atherosclerosis.

Topics: Aorta; Atherosclerosis; Catalase; Cells, Cultured; Chemokine CCL2; Coculture Techniques; Cytochalasin D; Cytokines; Endothelial Cells; Endothelium, Vascular; Humans; Inflammation Mediators; Interleukin-6; Interleukin-8; Microscopy, Confocal; Mouth; Streptococcus; Streptococcus anginosus; Streptococcus gordonii; Streptococcus intermedius; Streptococcus mitis; Streptococcus mutans; Streptococcus oralis; Viridans Streptococci; Virulence

The uptake of lipoproteins by macrophages is a critical step in the development of atherosclerotic lesions. Cultured monocyte-derived macrophages take up large amounts of native LDL by receptor-independent fluid-phase pinocytosis, either constitutively or in response to specific activating stimuli, depending on the macrophage phenotype. We therefore sought to determine whether fluid-phase pinocytosis occurs in vivo in macrophages in atherosclerotic lesions. We demonstrated that fluorescent pegylated nanoparticles similar in size to LDL (specifically nontargeted Qtracker quantum dot and AngioSPARK nanoparticles) can serve as models of LDL uptake by fluid-phase pinocytosis in cultured human monocyte-derived macrophages and mouse bone marrow-derived macrophages. Using fluorescence microscopy, we showed that atherosclerosis-prone Apoe-knockout mice injected with these nanoparticles displayed massive accumulation of the nanoparticles within CD68+ macrophages, including lipid-containing foam cells, in atherosclerotic lesions in the aortic arch. Similar results were obtained when atherosclerotic mouse aortas were cultured with nanoparticles in vitro. These results show that macrophages within atherosclerotic lesions can take up LDL-sized nanoparticles by fluid-phase pinocytosis and indicate that fluid-phase pinocytosis of LDL is a mechanism for macrophage foam cell formation in vivo.

Topics: Animals; Aorta; Aorta, Thoracic; Apolipoproteins E; Atherosclerosis; Cells, Cultured; Cholesterol, Dietary; Cytochalasin D; Female; Fluorescent Dyes; Foam Cells; Humans; Lipoproteins, LDL; Macrophages; Mice; Mice, Inbred C57BL; Mice, Knockout; Nanoparticles; Particle Size; Pinocytosis; Polyethylene Glycols; Quantum Dots