| Atherosclerosis is a chronic inflammatory disease of the arterial wall where both innate and adaptive immune responses contribute to disease initiation and progression. The role of the immune system in atherosclerosis has received considerable interest in recent years. However, sufficient knowledge to justify the immune modulatory mechanisms has not yet been obtained.Recent studies from several groups suggest that subtypes of T cells, called Tregs, previously shown to maintain immunological tolerance, inhibit the development and progression of atherosclerosis. Interestingly, the suppressive effects of these cells are not restricted to the adaptive immune system including CD4+T cells, CD8+T cells, natural killer (NK) cells, or B cells, but can also affect the activation and function of innate immune cells (monocytes, macrophages, dendritic cells) and neutrophils, but their effects on ECs are less well known.MicroRNAs are single-stranded, non-coding, small RNAs which regulate gene expression by destabilizing target mRNAs and/or translation inhibition. Recently, we identified that miR-181b inhibited NF-KB-mediated endothelial activation by reducing the expression of importin-3, a protein critical for NF-κB translocation from cytoplasm to nucleus. However, the role of miRNA-181b in the thrombin signaling in endothelial cells has not been examined.Here, we report that Tregs may exert their protective effects against atherogenesis in part through inducing an immune-inhibitory phenotype of ECs involving a CTLA-4-dependent cell-to-cell contact and also requiring soluble factors, by mainly IL-10and TGF-β; MiR-181b and its primary transcripts may be rapidly reduced by thrombin, but miR-181b could repress the cell adhesion molecules expression and leukocyte accumulation by impeding p65and importin-a3nuclear translocations as well as suppressing PI3K/Akt/IKKa/β signaling pathway, which is probably by directly targeting PIK3C2A. (Abstract) Part I. The role of CD4+CD25+Foxp3+regulatory T cells in protecting the activation and function of human umbilical vein endothelial cells impaired by ox-LDLObjective:The endothelial cells (ECs) play a major role in the atherogenic initiation, changing their quiescence into activated phenotypes to support every phase of the inflammatory process. The aim of this study is to investigate the role of CD4+CD25+Foxp3+T regulatory cells (Tregs) in protecting the activation and function of human umbilical vein endothelial cells (HUVECs) induced by pro-inflammatory stimulus and the mechanisms of it.Methods and Results:HUVECs were incubated alone, with Tregs or CD4+CD25-T cells in the presence of anti-CD3mAbs for48h, and then were stimulated with or without oxidized low density lipoprotein (ox-LDL)/lipopolysaccharide (LPS) for an additional24h. We show that Tregs are able to induce alternative expression of immune phenotypic markers of activated HUVECs by down-modulating CD86and to inhibit the adhesion molecule such as vascular cell adhesion molecule-1(VCAM-1), pro-inflammatory cytokines like monocyte chemoattractant protein-1(MCP-1) and interleukin-6(IL-6) response of HUVECs to ox-LDL/LPS. Moreover, Tregs down-regulate pro-inflammatory factor NF-κB activation and induce resistance to suppression of anti-inflammatory factor KLF2in HUVECs induced by pro-infla-mmatory stimulus. Mechanism studies reveal that Tregs-mediated suppression of HUVECs pro-inflammatory cytokines and adhesion molecule expression impaired by ox-LDL/LPS requires cell contact by cytotoxic T lymphocyte antigen-4(CTLA-4) and CD86as well as soluble factors, by mainly interleukin-10(IL-10) and transforming growth factor-β(TGF-0).Conclusions:Tregs may exert their protective effects against atherogenesis in part through inducing an immune-inhibitory phenotype of ECs involving a CTLA-4-dependent cell-to-cell contact and also requiring soluble factors, by mainly IL-10and TGF-β. (Abstract) Part Ⅱ. MicroRNA-181b inhibits thrombin-induced NF-kB signaling and endothelial cell activationObjective:Thrombin not only plays important role in mediating the cleavage of fibrinogen to fibrin, but also activates endothelial cells leading to a multitude of phenotypic changes like pereability, leukocyte trafficking, migration and hemostasis, etc. The aim of this study is to investigate the role of miR-181b in thrombin mediated NF-κB activation and the potential mechanisms of it.Methods and Results:HUVECs were treated with different concentration of thrombin for various of times espectively, then harvest for testing miR-181b and its precursors mir-181b1and mir-181b2expression, which were rapidly reduced by thrombin; HUVECs were transfected with miR-181b mimics or inhibitors for36hours, then treated with thrombin or combined with Calcein AM dyed THP-1cells for the indicated times, then did real-time q-PCR, western-blot or cell adhesion assays, we show that over expressing of miR-181b in HUVECs may repress the expression of cell adhesion molecules(CAMs) like VCAM-1, ICAM-1, E-selectin, as well as the leukocyte accumulation. Meanwhile, when transfected miR-181b inhibitor in HUVECs, the expression of CAMs and leukocyte accumulation were more lower than that in HUVCEs transfected with miR-181b inhibitor negative control. Moreover, we also show that mir-181b may impeding the nuclear of translocation of p65NF-κB and importin-a3in thrombin activated HUVECs. Signaling pathway studies reveal that miR-181b may repress the phosphorylation of the upstream genes in the NF-κB pathway, including IκBα, IKKa/β, p44/p42, p38, JNK and Akt in response to thrombin, which is probably by directly targeting PIK3C2A.Conclusions:MiR-181b and its primary transcripts are rapidly reduced by thrombin, miR-181b could repress the cell adhesion molecules expression and leukocyte accumulation by impeding p65and importin-a3nuclear translocations and suppressing PI3K/Akt/IKKa/β signaling pathway, which is probably by directly targeting PIK3C2A. |
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