Neutrophil extracellular traps modulate inflammatory markers and uptake of oxidized LDL by human and murine macrophages.

Neutrophil extracellular traps (NETs) are web-like structures, which are released upon neutrophil activation. It has previously been demonstrated that NETs are present in atherosclerotic lesions of both humans and animal models thus playing a decisive role in atherosclerosis. Besides, macrophages have a crucial role in disease progression, whereby classically activated M1 macrophages sustain inflammation and alternatively activated M2 macrophages display anti-inflammatory effects. Although NETs and macrophages were found to colocalize in atherosclerotic lesions, the impact of NETs on macrophage function is not fully understood. In the present study, we aimed to investigate the effect of NETs on human and murine macrophages in respect to the expression of pro-inflammatory cytokines, matrix metalloproteinases (MMPs) and uptake of oxidized LDL (oxLDL) in vitro. Human THP-1 and murine bone marrow-derived macrophages were cultured under M1 (LPS + IFN-γ)- and M2a (IL-4)-polarizing culture conditions and treated with NETs. To mimic intraplaque regions, cells were additionally cultured under hypoxic conditions. NETs significantly increased the expression of IL-1ß, TNF-α and IL-6 in THP-M1 macrophages under normoxia but suppressed their expression in murine M1 macrophages under hypoxic conditions. Notably, NETs increased the number of oxLDL-positive M1 and M2 human and murine macrophages under normoxia, but did not influence formation of murine foam cells under hypoxia. However, oxLDL uptake did not strongly correlate with the expression of the LDL receptor CD36. Besides, upregulated MMP-9 expression and secretion by macrophages was detected in the presence of NETs. Again, hypoxic culture conditions dampened NETs effects. These results suggest that NETs may favor foam cell formation and plaque vulnerability, but exert opposite effects in respect to the inflammatory response of human and murine M1 macrophages. Moreover, effects of NETs on macrophages' phenotype are altered under hypoxia.

MicroRNA-155 promotes the ox-LDL-induced activation of NLRP3 inflammasomes via the ERK1/2 pathway in THP-1 macrophages and aggravates atherosclerosis in ApoE-/- mice.

BACKGROUND: Nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome activation can induce the secretion of IL-1ß and IL-18 and after promoting the development of atherosclerosis. MiR-155 is an important microRNA that modulates inflammation in atherosclerosis, but the role of miR-155 in the regulation of the NLRP3 inflammasome is still unknown. METHODS: The atherosclerosis model was set up using ApoE-/- mice, and the lentiviral vector (LV) was used to interfere the expression of miR-155. HE stains was used for plaque morphology, immunohistochemistry (IHC) and western blot were used for protein expression quantification. We used oxidized low-density lipoprotein (ox-LDL) to incubate PMA-preprocessed THP-1 macrophages and detected NLRP3 inflammasome activation and ERK1/2 phosphorylation by western blot and Enzyme-linked immunosorbent assay. RESULTS: HE stains showed that the intravascular plaques in the miR-155-up group were remarkably increased, compared with negative control (NC) group. Results of IHC showed that the expression of caspase-1 and IL-1ß in the miR-155-up group was the highest of four groups, consist with the Western blot analysis. The results of in vitro experiment show that ox-LDL promoted NLRP3 inflammasome activation and ERK1/2 phosphorylation. Blocking the ERK1/2 pathway could inhibit ox-LDL-induced NLRP3 inflammasome activation. Moreover, we found that the overexpression of miR-155 promoted the activation of the ox-LDL-induced NLRP3 inflammasome, which could also be blocked by the ERK inhibitor U0126. CONCLUSIONS: MiR-155 aggravates the carotid AS lesion in ApoE-/- mice and exerts a regulatory effect on NLRP3 inflammasome activation in ox-LDL-induced macrophages via the ERK1/2 pathway.

Rheb (Ras Homolog Enriched in Brain 1) Deficiency in Mature Macrophages Prevents Atherosclerosis by Repressing Macrophage Proliferation, Inflammation, and Lipid Uptake.

OBJECTIVE: Macrophage foam cell formation is an important process in atherosclerotic plaque development. The small GTPase Rheb (Ras homolog enriched in brain 1) regulates endocytic trafficking that is critical for foam cell formation. However, it is unclear whether and how macrophage Rheb regulates atherogenesis, which are the focuses of the current study. Approach and Results: Immunofluorescence study confirmed the colocalization of Rheb in F4/80 and Mac-2 (galectin-3)-labeled lesional macrophages. Western blot and fluorescence-activated cell sorting analysis showed that Rheb expression was significantly increased in atherosclerotic lesions of atherosclerosis-prone (apoE-/- [apolipoprotein E deficient]) mice fed with Western diet. Increased Rheb expression was also observed in oxidized LDL (low-density lipoprotein)-treated macrophages. To investigate the in vivo role of macrophage Rheb, we established mature RhebmKO (macrophage-specific Rheb knockout) mice by crossing the Rheb floxed mice with F4/80-cre mice. Macrophage-specific knockout of Rheb in mice reduced Western diet-induced atherosclerotic lesion by 32%, accompanied with a decrease in macrophage content in plaque. Mechanistically, loss of Rheb in macrophages repressed oxidized LDL-induced lipid uptake, inflammation, and macrophage proliferation. On the contrary, lentivirus-mediated overexpression of Rheb in macrophages increased oxidized LDL-induced lipid uptake and inflammation, and the stimulatory effect of Rheb was suppressed by the mTOR (mammalian target of rapamycin) inhibitor rapamycin or the PKA (protein kinase A) activator forskolin. CONCLUSIONS: Macrophage Rheb plays important role in Western diet-induced atherosclerosis by promoting macrophage proliferation, inflammation, and lipid uptake. Inhibition of expression and function of Rheb in macrophages is beneficial to prevent diet-induced atherosclerosis.

The role of the mLDL-induced activation of the complement system classical pathway and C3 expression stimulation in atherosclerosis.

The role of modified low density lipoprotein in the activation of the classical pathway of the complement system and increasing expression C3 gene in human macrophages is described, role of these processes on the progression of atherosclerotic vascular lesions is considering.

The function of high-density lipoprotein and low-density lipoprotein in the maintenance of mouse ovarian steroid balance.

The membrane proteins, low-density lipoprotein receptor (LDLR) and scavenger receptor class B member 1 (SR-BI, gene name Scarb1), are lipoprotein receptors that play central roles in lipoprotein metabolism. Cholesterol bound in high-density lipoprotein (HDL) and LDL is transported into cells mainly by SR-BI and LDLR. The relative contribution of LDL and HDL to the steroidogenic cholesterol pool varies among species and may vary among tissues within one species. To investigate which of these pathways is more important in the supply of cholesterol in mouse ovary, we utilized immunohistochemistry, western blotting, RNAi, and RT-PCR as well as Ldlr-/- mice to explore the uptake of HDL and LDL in the ovary. Our data demonstrate that both SR-BI and LDLR are present in the interstitial cells, thecal cells, and corpora lutea (CLs), and their expression fluctuates with the development of follicles and CLs. The intracellular cholesterol concentration was significantly decreased when Ldlr or Scarb1 was silenced in luteal cells. Furthermore, Ldlr-/- mice had lower progesterone and estrogen levels compared to wild-type mice, and when Ldlr-/- mice were treated with the inhibitor of de novo cholesterol synthesis, lovastatin, serum progesterone, and estrogen concentrations were further reduced. These results demonstrate that both LDLR and SR-BI play important roles in importing cholesterol and that both HDL and LDL are crucial in steroidogenesis in mouse ovaries.

LDL switches the LRP6 internalization route from flotillin dependent to clathrin dependent in hepatic cells.

Low-density lipoprotein (LDL) receptor-related protein 6 (LRP6) was originally identified as a co-receptor of the Wnt signalling pathway and has been shown to be involved in LDL transport. In polarized hepatocytes, many apical proteins are sorted to the basolateral membrane and then internalized and transported to the apical bile canalicular membrane - a process known as transcytosis. We show that LRP6 is transcytosed to the apical membrane of polarized hepatic HepG2 cells via a flotillin-dependent manner in the absence of LDL. LRP6 formed a complex with Niemann-Pick type C1-like 1 (NPC1L1), which is localized to the bile canalicular membrane of the liver and is involved in cholesterol absorption from the bile. LRP6 was required for apical membrane localization of NPC1L1 in the absence of LDL. Clathrin-dependent LRP6 internalization occurred in the presence of LDL, which resulted in trafficking of LRP6 to the lysosome, thereby reducing apical sorting of LRP6 and NPC1L1. These results suggest that LRP6 endocytosis proceeds by two routes, depending on the presence of LDL, and that LRP6 controls the intracellular destination of NPC1L1 in hepatocytes.

miR-497 accelerates oxidized low-density lipoprotein-induced lipid accumulation in macrophages by repressing the expression of apelin.

microRNAs (miRNAs) play important roles in the pathogenesis of atherosclerosis. A previous study has reported that miR-497 is elevated in advanced atherosclerotic lesions in an apoE-deficient (apoE-/-) mouse model. The purpose of this study is to test whether miR-497 can modulate macrophage foam cell formation, an initiating event in atherosclerosis. We found that miR-497 was upregulated in THP-1 macrophages after treatment with oxidized low-density lipoprotein (oxLDL). Enforced expression of miR-497 promoted lipid accumulation and decreased cholesterol efflux in oxLDL-exposed THP-1 macrophages. In contrast, downregulation of miR-497 suppressed oxLDL-induced lipid accumulation in THP-1 macrophages. Apelin was identified to be a downstream target of miR-497. Overexpression of miR-497 significantly reduced the expression of apelin in THP-1 macrophages. Interestingly, delivery of a miR-497-resistant variant of apelin significantly inhibited lipid accumulation and enhanced cholesterol efflux in miR-497-overexpressing THP-1 macrophages in response to oxLDL. In addition, miR-497 expression was increased and negatively correlated with apelin protein expression in human atherosclerotic lesions. In conclusion, miR-497 contributes to oxLDL-induced lipid deposition in macrophages largely via targeting of apelin and thus represents a potential therapeutic target for atherosclerosis.

Oxidized low-density lipoproteins enhance expression and activity of CD39 and CD73 in the human aortic valve endothelium.

Extracellular nucleotides regulate thrombosis, inflammation, and immune response. Ectonucleoside triphosphate diphosphohydrolase 1 (CD39) and ecto-5'-nucleotidase (CD73) convert extracellular nucleotides in a sequential order: ATP to ADP, AMP, and then to adenosine. In this study, we aimed to test an effect of oxidized low-density lipoprotein (ox-LDL) on CD39 and CD73 in endothelial cells. Human aortic valve endothelial cells were exposed to ox-LDL for 24-48 h. Next, the activity, protein expression, and mRNA transcripts level of CD39 and CD73 were characterized by an incubation with ATP or AMP followed by high-performance liquid chromatography analysis of media as well as western blots and qPCR. CD73 activity in human valve endothelial cells was increased in presence of ox-LDL (4.04 ± 0.32 nmol/mg prot./min, mean +/- SEM) as compared with control (2.75 ± 0.21 nmol/mg prot/min). There was almost no effect of ox-LDL on CD39 activity. A similar effect was observed for mRNA and protein expression. In conclusion, we found that ox-LDL modulated CD39 and CD73 activity in the endothelium, which may contribute to relevant pathologies and featured treatments.

Endoplasmic Reticulum Stress Links Oxidative Stress to Impaired Pancreatic Beta-Cell Function Caused by Human Oxidized LDL.

Elevated plasma concentration of the pro-atherogenic oxidized low density lipoprotein cholesterol (LDL) triggers adverse effects in pancreatic beta-cells and is associated with type 2 diabetes. Here, we investigated whether the endoplasmic reticulum (ER) stress is a key player coupling oxidative stress to beta-cell dysfunction and death elicited by human oxidized LDL. We found that human oxidized LDL activates ER stress as evidenced by the activation of the inositol requiring 1α, and the elevated expression of both DDIT3 (also called CHOP) and DNAJC3 (also called P58IPK) ER stress markers in isolated human islets and the mouse insulin secreting MIN6 cells. Silencing of Chop and inhibition of ER stress markers by the chemical chaperone phenyl butyric acid (PBA) prevented cell death caused by oxidized LDL. Finally, we found that oxidative stress accounts for activation of ER stress markers induced by oxidized LDL. Induction of Chop/CHOP and p58IPK/P58IPK by oxidized LDL was mimicked by hydrogen peroxide and was blocked by co-treatment with the N-acetylcystein antioxidant. As a conclusion, the harmful effects of oxidized LDL in beta-cells requires ER stress activation in a manner that involves oxidative stress. This mechanism may account for impaired beta-cell function in diabetes and can be reversed by antioxidant treatment.

VNN1 promotes atherosclerosis progression in apoE-/- mice fed a high-fat/high-cholesterol diet.

Accumulated evidence shows that vanin-1 (VNN1) plays a key part in glucose metabolism. We explored the effect of VNN1 on cholesterol metabolism, inflammation, apoptosis in vitro, and progression of atherosclerotic plaques in apoE(-/-) mice. Oxidized LDL (Ox-LDL) significantly induced VNN1 expression through an ERK1/2/cyclooxygenase-2/PPARα signaling pathway. VNN1 significantly increased cellular cholesterol content and decreased apoAI and HDL-cholesterol (HDL-C)-mediated efflux by 25.16% and 23.13%, respectively, in THP-1 macrophage-derived foam cells (P < 0.05). In addition, VNN1 attenuated Ox-LDL-induced apoptosis through upregulation of expression of p53 by 59.15% and downregulation of expression of B-cell lymphoma-2 127.13% in THP-1 macrophage (P < 0.05). In vivo, apoE(-/-) mice were divided randomly into two groups and transduced with lentivirus (LV)-Mock or LV-VNN1 for 12 weeks. VNN1-treated mice showed increased liver lipid content and plasma levels of TG (124.48%), LDL-cholesterol (119.64%), TNF-α (148.74%), interleukin (IL)-1ß (131.81%), and IL-6 (156.51%), whereas plasma levels of HDL-C (25.75%) were decreased significantly (P < 0.05). Consistent with these data, development of atherosclerotic lesions was increased significantly upon infection of apoE(-/-) mice with LV-VNN1. These observations suggest that VNN1 may be a promising therapeutic candidate against atherosclerosis.

Lysosomes are involved in induction of steroidogenic acute regulatory protein (StAR) gene expression and progesterone synthesis through low-density lipoprotein in cultured bovine granulosa cells.

Progesterone is an important steroid hormone in the regulation of the bovine estrous cycle. The steroidogenic acute regulatory protein (StAR) is an indispensable component for transporting cholesterol to the inner mitochondrial membrane, which is one of the rate-limiting steps for progesterone synthesis. Low-density lipoprotein (LDL) supplies cholesterol precursors for progesterone formation, and the lysosomal degradation pathway of LDL is essential for progesterone biosynthesis in granulosa cells after ovulation. However, it is currently unknown how LDL and lysosomes coordinate the expression of the StAR gene and progesterone production in bovine granulosa cells. Here, we investigated the role of lysosomes in LDL-treated bovine granulosa cells. Our results reported that LDL induced expression of StAR messenger RNA and protein as well as expression of cholesterol side-chain cleavage cytochrome P-450 (CYP11A1) messenger RNA and progesterone production in cultured bovine granulosa cells. The number of lysosomes in the granulosa cells was also significantly increased by LDL; whereas the lysosomal inhibitor, chloroquine, strikingly abolished these LDL-induced effects. Our results indicate that LDL promotes StAR expression, synthesis of progesterone, and formation of lysosomes in bovine granulosa cells, and lysosomes participate in the process by releasing free cholesterol from hydrolyzed LDL.

Effect of Oxidized Low Density Lipoprotein on the Expression of Runx2 and SPARC Genes in Vascular Smooth Muscle Cells.

BACKGROUND: Vascular calcification is an important stage in atherosclerosis. During this stage, vascular smooth muscle cells (VSMC) synthesize many osteogenic factors such as osteonectin (encoded by SPARC). Oxidative stress plays a critical role in atherosclerosis progression, and its accumulation in the vascular wall stimulates the development of atherosclerosis and vascular calcification. The osteonectin overexpression has been observed in the arterial wall during the course of atherosclerosis. However, the regulatory mechanism of oxidized low density lipoprotein (oxLDL)-mediated vascular calcification remains to be clarified. The aim of this study was to investigate the effect of oxLDL on the osteonectin gene expression through the Runx2 transcription factor. METHODS: In this experimental study, VSMC were cultured in F-12K media and then treated with oxLDL. The expression of Runx2 and osteonectin genes was determined by real-time PCR method. Protein levels were investigated by the western blotting technique. The Runx2 gene was knocked down using siRNA in order to determine whether Runx2 regulates the osteonectin expression in VSMC induced by oxLDL. Then transfected cells were treated with oxLDL, and the expression levels of Runx2 and osteonectin were determined again. RESULTS: oxLDL was found to increase Runx2 and osteonectin gene expression (4.8±0.47- and 9.2±1.96-fold, respectively) after 48 h. Western blotting analysis confirmed the induced levels of Runx2 and osteonectin proteins. However, oxLDL-induced osteonectin expression was not observed to be blocked by Runx2 knockdown. CONCLUSION: The up-regulation of osteonectin by oxLDL is independent of Runx2, and it may be mediated by other transcription factors.

Activation of GPR55 Receptors Exacerbates oxLDL-Induced Lipid Accumulation and Inflammatory Responses, while Reducing Cholesterol Efflux from Human Macrophages.

The G protein-coupled receptor GPR55 has been proposed as a new cannabinoid receptor associated with bone remodelling, nervous system excitability, vascular homeostasis as well as in several pathophysiological conditions including obesity and cancer. However, its physiological role and underlying mechanism remain unclear. In the present work, we demonstrate for the first time its presence in human macrophages and its increased expression in ox-LDL-induced foam cells. In addition, pharmacological activation of GPR55 by its selective agonist O-1602 increased CD36- and SRB-I-mediated lipid accumulation and blocked cholesterol efflux by downregulating ATP-binding cassette (ABC) transporters ABCA1 and ABCG1, as well as enhanced cytokine- and pro-metalloprotease-9 (pro-MMP-9)-induced proinflammatory responses in foam cells. Treatment with cannabidiol, a selective antagonist of GPR55, counteracted these pro-atherogenic and proinflammatory O-1602-mediated effects. Our data suggest that GPR55 could play deleterious role in ox-LDL-induced foam cells and could be a novel pharmacological target to manage atherosclerosis and other related cardiovascular diseases.

A plant-based diet, atherogenesis, and coronary artery disease prevention.

A plant-based diet is increasingly becoming recognized as a healthier alternative to a diet laden with meat. Atherosclerosis associated with high dietary intake of meat, fat, and carbohydrates remains the leading cause of mortality in the US. This condition results from progressive damage to the endothelial cells lining the vascular system, including the heart, leading to endothelial dysfunction. In addition to genetic factors associated with endothelial dysfunction, many dietary and other lifestyle factors, such as tobacco use, high meat and fat intake, and oxidative stress, are implicated in atherogenesis. Polyphenols derived from dietary plant intake have protective effects on vascular endothelial cells, possibly as antioxidants that prevent the oxidation of low-density lipoprotein. Recently, metabolites of L-carnitine, such as trimethylamine-N-oxide, that result from ingestion of red meat have been identified as a potential predictive marker of coronary artery disease (CAD). Metabolism of L-carnitine by the intestinal microbiome is associated with atherosclerosis in omnivores but not in vegetarians, supporting CAD benefits of a plant-based diet. Trimethylamine-N-oxide may cause atherosclerosis via macrophage activation. We suggest that a shift toward a plant-based diet may confer protective effects against atherosclerotic CAD by increasing endothelial protective factors in the circulation while reducing factors that are injurious to endothelial cells. The relative ratio of protective factors to injurious endothelial exposure may be a novel approach to assessing an objective dietary benefit from a plant-based diet. This review provides a mechanistic perspective of the evidence for protection by a plant-based diet against atherosclerotic CAD.

Subcellular localization of DAXX influence ox-LDL induced apoptosis in macrophages.

Here we aimed to evaluate the effects of DAXX subcellular localization on ox-LDL induced macrophages apoptosis. Cytoplasmic localization vector DAXX-W621A and nuclear localization vector DAXX-S667A were constructed by point mutation in DAXX. Blank vector, full length DAXX, DAXX-W621A, DAXX-S667A was transfect into RAW264.7 cells, respectively. Then the cells were incubated with 100 mg/ml ox-LDL for 48 h. Immunofluorescent assay was used to assay the localization of DAXX. MTT and Flow cytometry was used to determine cellular viability and apoptosis. RT-PCR and Western blot were used to analyze the expression levels. A significantly increased expression of DAXX was found in transfected cells of DAXX. The content of DAXX in nucleus was significantly increased in DAXX(S667A), and DAXX was significantly increased in cytoplasm of DAXX(W621A). Besides, we found DAXX was mainly expressed in nucleus with a low-level expression in cytoplasm through immunofluorescence. However in DAXX(W621A) group, the DAXX began to transferred to cytoplasm, which exhibited significant florescence. After treated with ox-LDL, the cytoactive of DAXX-W621A exhibited significantly decreased level when compared DAXX group. However, after added inhibitor LMB, the inhibition was relieved. The cell viability was also significantly increased in DAXX-S667A group. The results of apoptosis rates were similar in each group. Furthermore, we found the expression of ASK1 and JNK was also consistent with the apoptosis rates. Cytoplasmic localization of DAXX can increase injury sensitivity of ox-LDL on cells, and nuclear localization can antagonise the effect of ox-LDL. Besides, it is certified ox-LDL induced apoptosis is mainly through ASK1-JNK pathway.

Resveratrol suppresses oxidised low-density lipoprotein-induced macrophage apoptosis through inhibition of intracellular reactive oxygen species generation, LOX-1, and the p38 MAPK pathway.

BACKGROUND/AIM: Resveratrol (RSV) may have therapeutic potential for various diseases. Here we investigated the effect of RSV on oxidised low-density lipoprotein- (ox-LDL) induced apoptosis in RAW264.7 macrophages. METHODS: Apoptosis of macrophages following incubation with ox-LDL (with or without RSV pre-treatment) was detected by flow cytometry. Western blotting was used to assess the protein expression of Bax, Bcl-2, and caspase-3 as well as ox-LDL receptor 1 (LOX-1) and p38 mitogen-activated protein kinase (MAPK) phosphorylation. Reactive oxygen species (ROS) generation was evaluated by 2', 7'-dichlorofluorescein diacetate, and JC-1 probe was used to determine the mitochondrial transmembrane potential of cells. RESULTS: Ox-LDL significantly reduced viability and increased the rate of apoptosis (P < 0.05) in RAW264.7 cells. However, pre-treatment with RSV resulted in a remarkable decrease in this apoptotic effect. Moreover, ox-LDL caused the up-regulation of Bax and the down-regulation of Bcl-2, as well as the activation of caspase-3. Expression of LOX-1, phosphorylation of p38 MAPK, and intracellular ROS production also increased after ox-LDL stimulation. Strikingly, these effects were abolished by pre-treatment of cells with RSV. CONCLUSION: RSV suppresses ox-LDL-induced macrophage apoptosis. These beneficial effects might be exerted through inhibition of ROS generation, LOX-1, and the p38 MAPK signalling pathway.

Dual effects of miR-155 on macrophages at different stages of atherosclerosis: LDL is the key?

Atherosclerosis is a chronic, maladaptive, nonresolving inflammatory response which underlies the leading cause of death in the world today. During the process, macrophages play a central role in both the initiation and development stages of disease pathogenesis. MicroRNAs are a class of small non-coding RNAs that regulate almost all biological processes. MiR-155 is multi-target molecule specifically expressed in atherosclerotic plaques and pro-inflammatory macrophages. However, the effects of miR-155 on atherogenesis have been controversial. Several lines of evidence collectively indicated that, both as inducers and carriers of miR-155, LDL and its oxidized derivatives could modulate miR-155-mediated inflammatory and apoptotic responses in lesional macrophages at different stages of atherosclerosis. During early lesion formation, both native and mildly-oxidized LDL facilitated endogenous miR-155-mediated macrophage activation and recruitment. In the meantime, they may also increase the accumulation of exogenous LDL-bound miR-155, along with lipid intake and foam cell formation. During advanced stages, the levels of exogenous miR-155 and extensively-oxidized LDL could gradually increase and become highly enough to synergistically induce macrophage apoptosis and atheroma formation. Taken together, we hypothesized that native LDL and oxidized LDL played a key role in modulating the effects of miR-155 on macrophages at different stages of atherosclerosis.

Cancer stem cell-specific scavenger receptor CD36 drives glioblastoma progression.

Glioblastoma (GBM) contains a self-renewing, tumorigenic cancer stem cell (CSC) population which contributes to tumor propagation and therapeutic resistance. While the tumor microenvironment is essential to CSC self-renewal, the mechanisms by which CSCs sense and respond to microenvironmental conditions are poorly understood. Scavenger receptors are a broad class of membrane receptors well characterized on immune cells and instrumental in sensing apoptotic cellular debris and modified lipids. Here, we provide evidence that CSCs selectively use the scavenger receptor CD36 to promote their maintenance using patient-derived CSCs and in vivo xenograft models. CD36 expression was observed in GBM cells in addition to previously described cell types including endothelial cells, macrophages, and microglia. CD36 was enriched in CSCs and was able to functionally distinguish self-renewing cells. CD36 was coexpressed with integrin alpha 6 and CD133, previously described CSC markers, and CD36 reduction resulted in concomitant loss of integrin alpha 6 expression, self-renewal, and tumor initiation capacity. We confirmed oxidized phospholipids, ligands of CD36, were present in GBM and found that the proliferation of CSCs, but not non-CSCs, increased with exposure to oxidized low-density lipoprotein. CD36 was an informative biomarker of malignancy and negatively correlated to patient prognosis. These results provide a paradigm for CSCs to thrive by the selective enhanced expression of scavenger receptors, providing survival, and metabolic advantages.

Toll-like receptor 4 mediates inflammatory cytokine secretion in smooth muscle cells induced by oxidized low-density lipoprotein.

Oxidized low-density lipoprotein (oxLDL)-regulated secretion of inflammatory cytokines in smooth muscle cells (SMCs) is regarded as an important step in the progression of atherosclerosis; however, its underlying mechanism remains unclear. This study investigated the role of toll-like receptor 4 (TLR4) in oxLDL-induced expression of inflammatory cytokines in SMCs both in vivo and in vitro. We found that the levels of TLR4, interleukin 1-ß (IL1-ß), tumor necrosis factor-α (TNFα), monocyte chemoattractant protein 1 (MCP-1) and matrix metalloproteinase-2 (MMP-2) expression were increased in the SMCs of atherosclerotic plaques in patients with femoral artery stenosis. In cultured primary arterial SMCs from wild type mice, oxLDL caused dose- and time-dependent increase in the expression levels of TLR4 and cytokines. These effects were significantly weakened in arterial SMCs derived from TLR4 knockout mice (TLR4-/-). Moreover, the secretion of inflammatory cytokines was blocked by TLR4-specific antibodies in primary SMCs. Ox-LDL induced activation of p38 and NFκB was also inhibited in TLR4-/- primary SMCs or when treated with TLR4-specific antibodies. These results demonstrated that TLR4 is a crucial mediator in oxLDL-induced inflammatory cytokine expression and secretion, and p38 and NFκB activation.

Ox-LDL induces ER stress and promotes the adipokines secretion in 3T3-L1 adipocytes.

Adipocytes behave as a rich source of adipokines, which may be the link between obesity and its complications. Endoplasmic reticulum (ER) stress in adipocytes can modulate adipokines secretion. The aim of this study is to evaluate the effect of oxidized low density lipoprotein (ox-LDL) treatment on ER stress and adipokines secretion in differentiated adipocytes. 3T3-L1 pre-adipocytes were cultured and differentiated into mature adipocytes in vitro. Differentiated adipocytes were incubated with various concentrations of ox-LDL (0-100 µg/ml) for 48 hours; 50 µg/ml ox-LDL for various times (0-48 hours) with or without tauroursodeoxycholic acid (TUDCA) (0-400 µM) pre-treatment. The protein expressions of ER stress markers, glucose regulated protein 78(GRP78) and CCAAT/enhancer binding protein [C/EBP] homologous protein (CHOP) in adipocytes were detected by Western blot. The mRNA expressions of visfatin and resistin were measured by real-time PCR and the protein release of visfatin and resistin in supernatant were determined by ELISA. Treatment with ox-LDL could increase the cholesterol concentration in adipocytes. Ox-LDL induced the expressions of GRP78 and CHOP protein in adipocytes and promoted visfatin and resistin secretion in culture medium in dose and time-dependent manner. TUDCA could attenuate the effect of ox-LDL on GRP78 and CHOP expressions and reduce visfatin and resistin at mRNA and protein level in dose-dependent manner. In conclusion, ox-LDL promoted the expression and secretion of visfatin and resistin through its activation of ER stress, which may be related to the increase of cholesterol load in adipocytes.