Xiumei Xie

Toll-like receptor 3 (TLR3) induces apoptosis via death receptors and mitochondria by up-regulating the transactivating p63 isoform α (tap63α)

Toll-like receptor 3 (TLR3), a member of the pathogen recognition receptors, is widely expressed in various cells and has been shown to activate immune signaling pathways by recognizing viral double-stranded RNA. Recently, it was reported that the activation of TLR3 induced apoptosis in some cells, but the detailed molecular mechanism is not fully understood. In this study, we found that in endothelial cells polyinosinic-polycytidylic acid (poly(I-C)) induced dose- and time-dependent cell apoptosis, which was elicited by TLR3 activation, as TLR3 neutralization and down-regulation repressed the apoptosis. Poly(I-C) induced the activation of both caspases 8 and 9, indicating that TLR3 triggered the signaling of both the extrinsic and intrinsic apoptotic pathways. Poly(I-C) up-regulated tumor necrosis factor-related apoptosis-inducing ligand and its receptors, death receptors 4/5, resulting in initiating the extrinsic pathway. Furthermore, poly(I-C) down-regulated anti-apoptotic protein, B cell lymphoma 2 (Bcl-2), and up-regulated Noxa, a key Bcl-2 homology 3-only antagonist of Bcl-2, leading to the priming of the intrinsic pathway. A p53-related protein, the transactivating p63 isoform α (TAp63α), was induced by TLR3 activation and contributed to the activation of both the intrinsic and extrinsic apoptotic pathways. Both the cells deficient in p63 gene expression by RNA interference and cells that overexpressed the N-terminally truncated p63 isoform α (ΔNp63α), a dominant-negative variant of TAp63α, by gene transfection, survived TLR3 activation. Taken together, TAp63α is a crucial regulator downstream of TLR3 to induce cell death via death receptors and mitochondria.

Systemic delivery of umbilical cord blood cells for stroke therapy: a review.

PURPOSE This review paper summarizes relevant studies, discusses potential mechanisms of transplanted cell-mediated neuroprotection, and builds a case for the need to establish outcome parameters that are critical for transplantation success. In particular, we outline the advantages and disadvantages of systemic delivery of human umbilical cord blood (HUCB) cells in the field of cellular transplantation for treating ischemic stroke. METHODS A MEDLINE/PubMed systematic search of published articles in peer-reviewed journals over the last 25 years was performed focusing on the theme of HUCB as donor graft source for transplantation therapy in neurological disorders with emphasis on stroke. RESULTS Ischemic stroke remains a leading cause of human death and disability. Although stroke survivors may gain spontaneous partial functional recovery, they often suffer from sensory-motor dysfunction, behavioral/neurological alterations, and various degrees of paralysis. Currently, limited clinical intervention is available to prevent ischemic damage and restore lost function in stroke victims. Stem cells from fetal tissues, bone marrow, and HUCB has emerged in the last few years as a potential cell transplant cell source for ischemic stroke, because of their capability to differentiate into multiple cell types and the possibility that they may provide trophic support for cell survival, tissue repair, and functional recovery. CONCLUSION A growing number of studies highlight the potential of systemic delivery of HUCB cells as a novel therapeutic approach for stroke. However, additional preclinical studies are warranted to reveal the optimal HUCB transplant regimen that is safe and efficacious prior to proceeding to large-scale clinical application of these cells for stroke therapy.

The role of profilin-1 in endothelial cell injury induced by advanced glycation end products (AGEs)

BackgroundAccumulation of advanced glycation end products (AGEs) in the vasculature triggers a series of morphological and functional changes contributing to endothelial hyperpermeability. The reorganisation and redistribution of the cytoskeleton regulated by profilin-1 mediates endothelial cell contraction, which results in vascular hyperpermeability. This study aimed to investigate the pivotal role of profilin-1 in the process of endothelial cell damage induced by AGEs.MethodsHuman umbilical vein endothelial cells (HUVECs) were incubated with AGEs. The mRNA and protein expression of profilin-1 was determined using real-time PCR and western blotting analyses. The levels of intercellular adhesion molecule-1 (ICAM-1), nitric oxide (NO) and reactive oxygen species (ROS), as well as the activities of nuclear factor-κB (NF-κB) and protein kinase C (PKC), were detected using the appropriate kits. The levels of asymmetric dimethylarginine (ADMA) were determined using HPLC. The distribution of the cytoskeleton was visualised using immunofluorescent staining.ResultsCompared with the control, incubation of endothelial cells with AGEs (200 μg/ml) for 4 or 24 h significantly up-regulated the mRNA and protein expression of profilin-1, markedly increased the levels of ICAM-1 and ADMA and decreased the production of NO (P<0.05, P<0.01), which was significantly attenuated by pretreatment with DPI (an antioxidant), GF 109203X (PKC inhibitor) or BAY-117082 (NF-κB inhibitor). DPI (10 μmol/L) markedly decreased the elevated levels of ROS induced by AGEs (200 μg/ml, 24 h); however, GF 109203X (10 μmol/L) and BAY-117082 (5 μmol/L) exhibited no significant effect on the formation of ROS by AGEs. Immunofluorescent staining indicated that AGEs markedly increased the expression of profilin-1 in the cytoplasm and the formation of actin stress fibres, resulting in the rearrangement and redistribution of the cytoskeleton. This effect was significantly ameliorated by DPI, GF 109203X, BAY-117082 or siRNA treatment of profilin-1. Incubation with DPI and GF 109203X markedly inhibited the activation of PKC triggered by AGEs, and DPI and BAY-117082 significantly decreased the activity of NF-κB mediated by AGEs. Disruption of profilin-1 gene expression attenuated the extent of endothelial abnormalities by reducing ICAM-1 and ADMA levels and elevating NO levels (P<0.05, P<0.01), but this disruption had no effect on the activities of NF-κB and PKC (P>0.05).ConclusionsThese findings suggested that profilin-1 might act as an ultimate and common cellular effector in the process of metabolic memory (endothelial abnormalities) mediated by AGEs via the ROS/PKC or ROS/NF-қB signalling pathways.

Extracellular nucleotide inhibits cell proliferation and negatively regulates Toll-like receptor 4 signalling in human progenitor endothelial cells

Extracellular nucleotides mediate a wide range of physiological effects by interacting with plasma membrane P2 purinergic receptors. P2 receptors are expressed in certain kinds of stem cells, and function to induce cytokine expression and to modulate cell proliferation. We have analysed the expression and the function of P2 receptors in human umbilical cord blood‐derived EPCs (endothelial progenitor cells). EPCs expressed P2X4,6,7 and P2Y2,4,11,13,14 receptors and extracellular ATP inhibited EPCs proliferation. As in a previous study, EPCs expressed functional TLR4 (Toll‐like receptor 4) and activation of TLR4 by LPS (lipopolysaccharide) evoked a pro‐inflammatory immune response. When human EPCs were stimulated with LPS and nucleotides, ATP or UTP inhibited the expression of pro‐inflammatory cytokines including MCP‐1 (monocyte chemoattractant protein‐1), IFNα (interferon α), TNFα (tumour necrosis factor α) and adhesion molecule VCAM‐1 (vascular cell adhesion molecule 1) induced by LPS. ATP and UTP also down‐regulated the gene expression of TLR4, CD14 and MyD88 (myeloid differentiation factor 88), a TLR adaptor molecule, and protein expression of CD14 and MyD88. Moreover, the phosphorylation of NF‐κB (nuclear factor κB) p65 induced by TLR4 activation was inhibited partly by ATP or UTP at concentrations of 1–5 μM. These results suggest that extracellular nucleotides negatively regulate EPCs proliferation and TLR4 signalling.

The functional expression of TLR3 in EPCs impairs cell proliferation by induction of cell apoptosis and cell cycle progress inhibition

Toll-like receptor 3 (TLR3), a member of the TLR family that recognizes double-stranded RNA (dsRNA), plays an important role in antiviral immunity. TLR3 is widely expressed in various cells and the activation of TLR3 induces cell apoptosis in some cells. However, the effect of TLR3 on cell proliferation in endothelial progenitor cells (EPCs) is unclear. In this study, we found that EPCs expressed high levels of TLR1, 3, 4, and 6 and low levels of TLR2, 5, 7, 8, and 10. The treatment of EPCs with TLR3 agonist Poly I:C up-regulated the expression of cytokines IL-1β, IL-6, IL-8, TNF-α, IFN-α, and IFN-β, indicating that EPCs expressed functional TLR3. Moreover, Poly I:C treatment induced cell cycle progress inhibition and cell apoptosis, leading to the inhibition of cell proliferation. Further studies indicated that IL-1β was involved in TLR3-induced cell proliferation inhibition, as IL-1β inhibited cell proliferation in a dose-dependent manner, and the IL-1β receptor type I (IL-1R1)-neutralizing antibody ameliorated Poly I:C-induced cell proliferation inhibition. Taken together, these results suggest that Poly I:C impairs cell proliferation by inducing cell cycle progress inhibition and cell apoptosis via TLR3 in EPCs.

The effect of endothelial progenitor cells on angiotensin II-induced proliferation of cultured rat vascular smooth muscle cells.

Abstract Previous studies have demonstrated that endothelial progenitor cells (EPCs) could delay the progress of vascular remodeling in blood vessel–proliferating diseases. The proliferation of vascular smooth muscle cells (VSMCs) is a pivotal factor in cardiovascular diseases. In this study, we investigated whether EPCs could inhibit the Angiotensin II (Ang II)–induced proliferation of VSMCs. The effect of early EPC-conditioned medium (E-EPC-CM), late EPCs-CM (L-EPC-CM), and HUVEC-CM on Ang II–induced proliferation of VSMCs was assessed by BrdU incorporation, total protein content, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays, and flow cytometry. Reverse transcriptase–polymerase chain reaction and Western blot were performed to analyze the effect of different CMs on Ang II–induced phosphorylations of ERK, JNK, p38, and NF-&kgr;B subunit p65 and the expressions of c-myc and c-fos. E-EPC-CM, L-EPC-CM, and HUVEC-CM significantly inhibited the Ang II–induced DNA synthesis, total protein expression, cell survival, and cell cycle progress of VSMCs. Furthermore, E-EPC-CM significantly inhibited the Ang II–induced phosphorylation of ERK, JNK, p38, and p65 (nuclear translocation of p65) and the expressions of c-myc and c-fos. Taken together, these data suggested that EPCs may delay the progress of vascular remodeling in blood vessel–proliferating diseases by inhibiting Ang II–induced proliferation of VSMCs through inactivating MAPKs and NF-&kgr;B signaling pathways and by reducing the expressions of c-myc and c-fos.

TNF-α-induced NOD2 and RIP2 contribute to the up-regulation of cytokines induced by MDP in monocytic THP-1 cells†

Nucleotide‐binding oligomerization domain containing 2 (NOD2)‐induced signal transduction and cytokine production is regulated by a number of factors. However, the feedback effect of the pro‐inflammatory TNF‐α on NOD2‐induced inflammation is not fully understood. In this study, we found unexpectedly that TNF‐α up‐regulated NOD2 ligand MDP‐induced production of the CXC chemokines, including CXCL1, 2, and 8, and the pro‐inflammatory cytokines, including IL‐1β, IL‐6, and TNF‐α, in a dose‐dependent manner at both mRNA and protein levels in monocytic THP‐1 cells. Though TNF‐α induced the up‐regulation of ubiquitin‐editing enzyme A20, an important negative regulator for Toll‐like receptor‐ and NOD2‐induced inflammatory responses, the over‐expression of A20 by gene transfer did not reversed MDP‐induced production of cytokines, suggested that A20 did not regulate the functions of NOD2 in THP‐1 cells. Meanwhile, we found that TNF‐α up‐regulated NOD2 and its down‐stream adaptor protein RIP2 at both mRNA and protein levels. MDP induced the activation of ERK, JNK, p38 and NF‐κB, and TNF‐α pre‐treatment augmented this activation. The results from pharmacological inhibition assay showed that cytokine production was dependent on MAPK signaling. In addition, we found that the pre‐treatment of THP‐1 cells with MDP down‐regulated the mRNA levels of cytokine induced by MDP re‐treatment. MDP pre‐treatment up‐regulated NOD2, but down‐regulated RIP2, and down‐regulated NOD2 signal transduction induced by MDP re‐stimulation. Taking together, these results suggested that TNF‐α is a positive regulator for NOD2 functions via up‐regulation of NOD2 and its signal adaptor RIP2, and TNF‐α‐induced A20 does not regulate MDP‐induced inflammatory responses in THP‐1 cells. J. Cell. Biochem. 119: 5072–5081, 2018.

MDP Up-Regulates the Gene Expression of Type I Interferons in Human Aortic Endothelial Cells

Muramyldipeptide (MDP), the minimum essential structure responsible for the immuno-adjuvant activity of peptidoglycan, is recognized by intracellular nuclear-binding oligomerization domain 2 (NOD2). Here, we obtained evidence that the treatment of human aortic endothelial cells (HAECs) with MDP up-regulated the gene expression of type I interferons in a dose- and time-dependent manner. MDP also up-regulated the expression of the receptor NOD2, suggesting that MDP may induce a positive feedback response. The up-regulation of interferons was not dependent on the TNFα signaling, as HAECs did not express TNFα with the stimulation of MDP, and TNFα neutralizing antibody did not decrease the induction of IFNs induced by MDP. RT-PCR results showed that HAECs expressed the gene transcripts of interferon regulatory factor (IRF) 1, 2, 3, 9. The western blot results showed that MDP induced the phosphorylation of IRF3. These results suggested that MDP induced the up-regulation of gene transcript of interferons through the activation of IRF3 signaling pathway. Meanwhile, MDP induced the gene expression of pro-inflammatory cytokines, including IL-1β, IL-8, and MCP-1. Taken together, these results suggested that HAECs may play roles in the anti-infection immune response and in the induction of innate immunity.

The suppression of ox-LDL-induced inflammatory cytokine release and apoptosis of HCAECs by long non-coding RNA-MALAT1 via regulating microRNA-155/SOCS1 pathway

BACKGROUND Atherosclerosis is a chronic inflammatory disease. Accumulating evidence suggests that long non-coding RNAs (lncRNAs) and microRNAs have emerged as critical regulators of atherosclerosis; however, whether they have crosstalk on this issue remains elusive. Here, we investigated the potential associations between lncRNA-MALAT1 and miR-155 on the regulation of atherosclerosis. METHODS Quantitative real-time PCR was employed to assess the expression of MALAT1, IL-6 and IL-8. ELISA was performed to measure the secretion of IL-6 and IL-8. MTT assay was used to determine the proliferation of Human Coronary Artery Endothelial Cells (HCAECs). Flow cytometry was used to measure the cell apoptosis. Western blot was used to assess the expression of apoptosis-related proteins and the phosphorylation of STAT1 and STAT3. RESULTS We found that the pro-inflammatory cytokine release and the apoptosis of HCAECs were elevated upon ox-LDL treatment, while MALAT1 expression was also up regulated. Knocking down of MALAT1 boosted ox-LDL-induced cytokine release and apoptosis of HCAECs. The binding site of miR-155 in MALAT1 sequence was confirmed by dual luciferase assay. Furthermore, miR-155 inhibition significantly repressed ox-LDL mediated inflammation and apoptosis of HCAECs via SOCS1. At last, we found that MALAT1 could suppress the inflammatory cytokine release and cell apoptosis via sponging miR-155 to increase SOCS1 level, which in turn restrained JAK-STAT pathway. CONCLUSION In summary, this study revealed the mechanisms by which MALAT1 worked as a putative atherosclerosis suppressor via miR-155 and SOCS1. Therefore, modulation of MALAT1/miR-155/SOCS1 axis might alleviate the inflammation persisted in atherosclerosis.