Total Flavonoids of Engelhardia roxburghiana Wall. Leaves Alleviated Foam Cells Formation through AKT/mTOR-Mediated Autophagy in the Progression of Atherosclerosis

Engelhardia roxburghiana Wall. is a traditional Chinese medicine used for treating cardiovascular diseases. Our previous study has implicated potential effects of total flavonoids of Engelhardia roxburghiana Wall. (TFER) against hyperlipidemia. The aim of the study is to uncover the effects and underlying mechanisms of TFER on foam cells formation after atherosclerosis. We used high fat diet (HFD) induced Apoe-/- mice and oxidized density lipoprotein (ox-LDL) induced THP-1 cells to mimic process of atherosclerosis in vivo and in vitro, respectively. Lipid accumulation, inflammation response, autophagosomes formation and expressions of autophagy related target genes were assessed. Our present study demonstrated TFER (500 mg/kg) alleviated macrophage infiltration and lipid accumulation in thoracic aortas of HFD-treated mice. In ox-LDL-treated THP-1 cells, MDC staining and Western blot analysis all indicated that the TFER (200 μg/ml) reduced foam cells formation and IL-1β releasing, activated autophagy through suppressing AKT/mTOR signaling, significantly regulating expressions of AKT, p-AKT, mTOR, p-mTOR, Beclin 1, LC3-II, p62. It is suggested that TFER alleviated atherosclerosis progression in vivo and in vitro through reducing foam cells formation and inflammatory responses, and the possible mechanism may be due to the activation of macrophage autophagy by inhibiting AKT and mTOR phosphorylation.     

Dose-specific effects of tumor necrosis factor alpha on osteogenic differentiation of mesenchymal stem cells

Objectives: To investigate tumor necrosis factor alpha (TNF‐α)‐induced changes in osteogenic differentiation from mesenchymal stem cells (MSCs). Materials and methods: Blockade of nuclear factor‐κB (NF‐κB) was achieved in ST2 murine MSCs via overexpression of the NF‐κB inhibitor, IκBα. Osteogenic differentiation was induced in IκBα‐overexpressing ST2 cells and normal ST2 cells when these cells were treated with TNF‐α at various concentrations. Expression levels of bone marker genes were determined using real time RT‐PCR and ALP activity assay. In vitro mineralization was performed to determine long‐term exposure to TNF‐α on mineral nodule formation. MTT assay was used to determine the changes in cell proliferation/survival. Results: Levels of Runx2, Osx, OC and ALP were up‐regulated in cell cultures treated with TNF‐α at lower concentrations, while down‐regulated in cell cultures treated with TNF‐α at higher concentrations. Blockade of NF‐κB signaling reversed the inhibitory effect observed in cell cultures treated with TNF‐α at higher concentrations, but showed no effect on cell cultures treated with TNF‐α at lower concentrations. In contrast, long‐term treatment of TNF‐α at all concentrations induced inhibitory effects on in vitro mineral nodule formation. MTT assay showed that TNF‐α inhibits proliferation/survival of mesenchymal stem cells when the NF‐κB signaling pathway is blocked. Conclusions: The binding of TNF‐α to its receptors results in the activation of multiple signaling pathways, which actively interact with each other to regulate the differentiation, proliferation, survival and apoptosis of MSCs.     

NDR1/STK38 potentiates NF‐κB activation by its kinase activity

Human NDR1/STK38 belongs to the nuclear‐Dbf2‐related (NDR) family of Ser/Thr kinases. It has been implicated to function in centrosome duplication, control of cell cycle and apoptosis. However, the mechanism of NDR1 signaling pathway remains largely elusive. Here, we report a novel role of NDR1 in NF‐κB activation. By overexpression, NDR1 potentiates NF‐κB activation induced by TNFα, whereas knockdown of NDR1 expression inhibits NF‐κB activation induced by TNFα. Coimmunoprecipitation shows that NDR1 interacts with multiple signal components except p65 in NF‐κB signaling pathway. Furthermore, both phosphorylation and kinase dead mutants of NDR1 lose their synergistic effects on TNFα‐induced NF‐κB activation. siRNA oligo against NDR1 and kinase dead mutant as well mainly block the NF‐κB activation induced by TRAF2 but not RIP1. Furthermore, kinase dead mutant of NDR1 fails to interact with TRAF2. Taken together, our findings suggest an unknown function of NDR1, which may regulate NF‐κB activation by its kinase activity. Copyright © 2012 John Wiley & Sons, Ltd.     

Autophagy is required for the activation of NFκB

It is well-established that the activation of the inhibitor of NFκB (IκBα) kinase (IKK) complex is required for autophagy induction by multiple stimuli. Here, we show that in autophagy-competent mouse embryonic fibroblasts (MEFs), distinct autophagic triggers, including starvation, mTOR inhibition with rapamycin and p53 inhibition with cyclic pifithrin α lead to the activation of IKK, followed by the phosphorylation-dependent degradation of IκBα and nuclear translocation of NFκB. Remarkably, the NFκB signaling pathway was blocked in MEFs lacking either the essential autophagy genes Atg5 or Atg7. In addition, we found that tumor necrosis factor α (TNFα)-induced NFκB nuclear translocation is abolished in both Atg5- and Atg7-deficient MEFs. Similarly, the depletion of essential autophagy modulators, including ATG5, ATG7, Beclin 1 and VPS34, by RNA interference inhibited TNFα-driven NFκB activation in two human cancer cell lines. In conclusion, it appears that, at least in some instances, autophagy is required for NFκB activation, highlighting an intimate crosstalk between these two stress response signaling pathways.     

Exosomes derived from mature dendritic cells increase endothelial inflammation and atherosclerosis via membrane TNF‐α mediated NF‐κB pathway

Whether dendritic cell (DC) derived exosomes play a role in the progression of endothelial inflammation and atherosclerosis remains unclear. Using a transwell system and exosome release inhibitor GW4869, we demonstrated that mature DCs contributed to endothelial inflammation and exosomes were involved in the process. To further confirm this finding, we isolated exosomes from bone marrow dendritic cell (BMDC) culture medium (named DC‐exos) and stimulated human umbilical vein endothelial cell (HUVEC) with these DC‐exos. We observed that mature DC‐exos increased HUVEC inflammation through NF‐κB pathway in a manner similar to that of lipopolysaccharide. After a protein array analysis of exosomes, we identified and confirmed tumour necrosis factor (TNF)‐α on exosome membrane being the trigger of NF‐κB pathway in HUVECs. We then performed an in vivo study and found that the aorta endothelial of mice could uptake intravenously injected exosomes and was activated by these exosomes. After a period of 12 weeks of mature DC‐exos injection into ApoE?/? mice, the atherosclerotic lesions significantly increased. Our study demonstrates that mature DCs derived exosomes increase endothelial inflammation and atherosclerosis via membrane TNF‐α mediated NF‐κB pathway. This finding extends our knowledge on how DCs affect inflammation and provides a potential method to prevent endothelial inflammation and atherosclerosis.     

A TNF‐p100 pathway subverts noncanonical NF‐κB signaling in inflamed secondary lymphoid organs

Lymphotoxin‐beta receptor (LTβR) present on stromal cells engages the noncanonical NF‐κB pathway to mediate RelB‐dependent expressions of homeostatic chemokines, which direct steady‐state ingress of naïve lymphocytes to secondary lymphoid organs (SLOs). In this pathway, NIK promotes partial proteolysis of p100 into p52 that induces nuclear translocation of the RelB NF‐κB heterodimers. Microbial infections often deplete homeostatic chemokines; it is thought that infection‐inflicted destruction of stromal cells results in the downregulation of these chemokines. Whether inflammation per se also regulates these processes remains unclear. We show that TNF accumulated upon non‐infectious immunization of mice similarly downregulates the expressions of these chemokines and consequently diminishes the ingress of naïve lymphocytes in inflamed SLOs. Mechanistically, TNF inactivated NIK in LTβR‐stimulated cells and induced the synthesis of Nfkb2 mRNA encoding p100; these together potently accumulated unprocessed p100, which attenuated the RelB activity as inhibitory IκBδ. Finally, a lack of p100 alleviated these TNF‐mediated inhibitions in inflamed SLOs of immunized Nfkb2^?/? mice. In sum, we reveal that an inhibitory TNF‐p100 pathway modulates the adaptive compartment during immune responses.     

Relation between TLR4/NF‐κB signaling pathway activation by 27‐hydroxycholesterol and 4‐hydroxynonenal,and atherosclerotic plaque instability

It is now thought that atherosclerosis, although due to increased plasma lipids, is mainly the consequence of a complicated inflammatory process, with immune responses at the different stages of plaque development. Increasing evidence points to a significant role of Toll‐like receptor 4 (TLR4), a key player in innate immunity, in the pathogenesis of atherosclerosis. This study aimed to determine the effects on TLR4 activation of two reactive oxidized lipids carried by oxidized low‐density lipoproteins, the oxysterol 27‐hydroxycholesterol (27‐OH) and the aldehyde 4‐hydroxynonenal (HNE), both of which accumulate in atherosclerotic plaques and play a key role in the pathogenesis of atherosclerosis. Secondarily, it examined their potential involvement in mediating inflammation and extracellular matrix degradation, the hallmarks of high‐risk atherosclerotic unstable plaques. In human promonocytic U937 cells, both 27‐OH and HNE were found to enhance cell release of IL‐8, IL‐1β, and TNF‐α and to upregulate matrix metalloproteinase‐9 (MMP‐9) via TLR4/NF‐κB‐dependent pathway; these actions may sustain the inflammatory response and matrix degradation that lead to atherosclerotic plaque instability and to their rupture. Using specific antibodies, it was also demonstrated that these inflammatory cytokines increase MMP‐9 upregulation, thus enhancing the release of this matrix‐degrading enzyme by macrophage cells and contributing to plaque instability. These innovative results suggest that, by accumulating in atherosclerotic plaques, the two oxidized lipids may contribute to plaque instability and rupture. They appear to do so by sustaining the release of inflammatory molecules and MMP‐9 by inflammatory and immune cells, for example, macrophages, through activation of TLR4 and its NF‐κB downstream signaling.     

The TGFβ1 pathway is required for NFκB dependent gene expression in mouse keratinocytes

The transforming growth factor-beta 1 (TGFβ1) and NFκB pathways are important regulators of epidermal homeostasis, inflammatory responses and carcinogenesis. Previous studies have shown extensive crosstalk between these pathways that is cell type and context dependent, but this has not been well-characterized in epidermal keratinocytes. Here we show that in primary mouse keratinocytes, TGFβ1 induces NFκB-luciferase reporter activity that is dependent on both NFκB and Smad3. TGFβ1-induced NFκB-luciferase activity was blocked by the IκB inhibitor parthenolide, the IκB super-repressor, a dominant negative TGFβ1-activated kinase 1 (TAK1) and genetic deletion of NFκB1. Coexpression of NFκB p50 or p65 subunits enhanced NFκB-luciferase activity. Similarly, inhibition of the TGFβ1 type I receptor with SB431542 or genetic deletion of Smad3 blocked TGFβ1 induction of NFκB-luciferase. TGFβ1 rapidly induced IKK phosphorylation but did not cause a detectable decrease in cytoplasmic IκB levels or nuclear translocation of NFκB subunits, although EMSA showed rapid NFκB nuclear binding activity that could be blocked by SB431542 treatment. TNFα, a well characterized NFκB target gene was also induced by TGFβ1 and this was blocked in NFκB+/− and −/− keratinocytes and by the IκB super-repressor. To test the effects of the TGFβ1 pathway on a biologically relevant activator of NFκB, we exposed mice and primary keratinocytes in culture to UVB irradiation. In primary keratinocytes UVB caused a detectable increase in levels of Smad2 phosphorylation that was dependent on ALK5, but no significant increase in SBE-dependent gene expression. Inhibition of TGFβ1 signaling in primary keratinocytes with SB431542 or genetic deletion of Tgfb1 or Smad3 suppressed UVB induction of TNFα message. Similarly, UVB induction of TNFα mRNA was blocked in skin of Tgfb1+/− mice. These studies demonstrate that intact TGFβ1 signaling is required for NFκB-dependent gene expression in mouse keratinocytes and skin and suggest that a convergence of these pathways in the nucleus rather than the cytoplasm may be critical for regulation of inflammatory pathways in skin by TGFβ1.     

FAK activation is required for TNF‐α‐induced IL‐6 production in myoblasts

Tumor necrosis factor‐α (TNF‐α) is a pleiotropic cytokine produced by activated macrophages. IL‐6 is a multifunctional cytokine that plays a central role in both innate and acquired immune responses. We investigated the signaling pathway involved in IL‐6 production stimulated by TNF‐α in cultured myoblasts. TNF‐α caused concentration‐dependent increases in IL‐6 production. TNF‐α‐mediated IL‐6 production was attenuated by focal adhesion kinase (FAK) mutant and siRNA. Pretreatment with phosphatidylinositol 3‐kinase inhibitor (PI3K; Ly294002 and wortmannin), Akt inhibitor, NF‐κB inhibitor (pyrrolidine dithiocarbamate, PDTC), and IκB protease inhibitor (L ‐1‐tosylamido‐2‐phenyl phenylethyl chloromethyl ketone, TPCK) also inhibited the potentiating action of TNF‐α. TNF‐α increased the FAK, PI3K, and Akt phosphorylation. Stimulation of myoblasts with TNF‐α activated IκB kinase α/β (IKKα/β), IκBα phosphorylation, p65 phosphorylation, and κB‐luciferase activity. TNF‐α mediated an increase of κB‐luciferase activity which was inhibited by Ly294002, wortmannin, Akt inhibitor, PDTC and TPCK or FAK, PI3K, and Akt mutant. Our results suggest that TNF‐α increased IL‐6 production in myoblasts via the FAK/PI3K/Akt and NF‐κB signaling pathway. J. Cell. Physiol. 223: 389–396, 2010. © 2010 Wiley‐Liss, Inc.     

Short‐Term Lenalidomide (Revlimid) Administration Ameliorates Cardiomyocyte Contractile Dysfunction in ob/ob Obese Mice

Lenalidomide is a potent immunomodulatory agent capable of downregulating proinflammatory cytokines such as tumor necrosis factor‐α (TNF‐α) and upregulating anti‐inflammatory cytokines. Lenalidomide has been shown to elicit cardiovascular effects, although its impact on cardiac function remains obscure. This study was designed to examine the effect of lenalidomide on cardiac contractile function in ob/ob obese mice. C57BL lean and ob/ob obese mice were given lenalidomide (50 mg/kg/day, p.o.) for 3 days. Body fat composition was assessed by dual‐energy X‐ray absorptiometry. Cardiomyocyte contractile and intracellular Ca²⁺ properties were evaluated. Expression of TNF‐α, interleukin‐6 (IL‐6), Fas, Fas ligand (FasL), the short‐chain fatty acid receptor GPR41, the NFκB regulator IκB, endoplasmic reticulum (ER) stress, the apoptotic protein markers Bax, Bcl‐2, caspase‐8, tBid, cytosolic cytochrome C, and caspase‐12; and the stress signaling molecules p38 and extracellular signal‐regulated kinase (ERK) were evaluated by western blot. ob/ob mice displayed elevated serum TNF‐α and IL‐6 levels, fat composition and glucose intolerance, the effects of which except glucose intolerance and fat composition were attenuated by lenalidomide. Cardiomyocytes from ob/ob mice exhibited depressed peak shortening (PS) and maximal velocity of shortening/relengthening, prolonged time‐to‐PS and time‐to‐90% relengthening as well as intracellular Ca²⁺ mishandling, which were ablated by lenalidomide. Western blot analysis revealed elevated levels of TNF‐α, IL‐6, Fas, Bip, Bax, caspase‐8, tBid, cleaved caspase‐3 caspase‐12, cytochrome C, phosphorylation of p38, and ERK in ob/ob mouse hearts, the effects of which with the exception of Bip, Bax, and caspase‐12 were alleviated by lenalidomide. Taken together, these data suggest that lenalidomide is protective against obesity‐induced cardiomyopathy possibly through antagonism of cytokine/Fas‐induced activation of stress signaling and apoptosis.     

Endothelial primary cilia inhibit atherosclerosis

Primary cilia are microtubule‐based structures present on most mammalian cells that are important for intercellular signaling. Cilia are present on a subset of endothelial cells where they project into the vessel lumen and are implicated as mechanical sensors of blood flow. To test the in vivo role of endothelial cilia, we conditionally deleted Ift88, a gene required for ciliogenesis, in endothelial cells of mice. We found that endothelial primary cilia were dispensable for mammalian vascular development. Cilia were not uniformly distributed in the mouse aorta, but were enriched at vascular branch points and sites of high curvature. These same sites are predisposed to the development of atherosclerotic plaques, prompting us to investigate whether cilia participate in atherosclerosis. Removing endothelial cilia increased atherosclerosis in Apoe^?/? mice fed a high‐fat, high‐cholesterol diet, indicating that cilia protect against atherosclerosis. Removing endothelial cilia increased inflammatory gene expression and decreased eNOS activity, indicating that endothelial cilia inhibit pro‐atherosclerotic signaling in the aorta.