BACKGROUND: NDRG-1 (N-myc downstream-regulated gene 1) is a member of NDRG family that plays essential roles in cell differentiation, proliferation, and stress responses. Although the expression of NDRG1 is regulated by fluid shear stress, its roles in vascular biology remain poorly understood. The purpose of the study is to determine the functional significance of NDRG1 in vascular inflammation and remodeling. METHODS AND RESULTS: By using quantitative polymerase chain reaction, western blot, and immunohistochemistry, we demonstrate that the expression of NDRG1 is markedly increased in cytokine-stimulated endothelial cells and in human and mouse atherosclerotic lesions. To determine the role of NDRG1 in endothelial activation, we performed loss-of-function studies using NDRG1 short hairpin RNA. Our results demonstrate that NDRG1 knockdown by lentivirus bearing NDRG1 short hairpin RNA substantially attenuates both IL-1β and TNF-α (tumor necrosis factor-α)-induced expression of cytokines/chemokines and adhesion molecules. Intriguingly, inhibition of NDRG1 also significantly attenuates the expression of procoagulant molecules, such as PAI-1 (plasminogen activator inhibitor type 1) and TF (tissue factor), and increases the expression of TM (thrombomodulin) and t-PA (tissue-type plasminogen activator), thus exerting potent antithrombotic effects in endothelial cells. Mechanistically, we showed that NDRG1 interacts with orphan Nur77 (nuclear receptor) and functionally inhibits the transcriptional activity of Nur77 and NF-κB (nuclear factor Kappa B) in endothelial cells. Moreover, in NDRG1 knockdown cells, both cytokine-induced mitogen-activated protein kinase activation, c-Jun phosphorylation, and AP-1 (activator protein 1) transcriptional activity are substantially inhibited. Neointima and atherosclerosis formation induced by carotid artery ligation and arterial thrombosis were markedly attenuated in endothelial cell-specific NDRG1 knockout mice compared with their wild-type littermates. CONCLUSIONS: Our results for the first time identify NDRG1 as a critical mediator implicated in regulating endothelial inflammation, thrombotic responses, and vascular remodeling, and suggest that inhibition of NDRG1 may represent a novel therapeutic strategy for inflammatory vascular diseases, such as atherothrombosis and restenosis.
Bovine viral diarrhea virus (BVDV) is the causative agent of bovine viral diarrhea-mucosal disease (BVD-MD), an important viral disease in cattle that is responsible for extensive economic losses to the cattle industry worldwide. Currently, several underlying mechanisms involved in viral replication, pathogenesis, and evading host innate immunity of BVDV remain to be elucidated, particularly during the early stage of virus infection. To further explore the mechanisms of BVDV-host interactions, the transcriptomics and proteomics profiles of BVDV-infected MDBK cells were sequenced using RNA-seq and iTRAQ techniques, respectively, and followed by an integrative analysis. Compared with mock-infected MDBK cells, a total of 665 differentially expressed genes (DEGs) (391 down-regulated, 274 up-regulated) and 725 differentially expressed proteins (DEPs) (461 down-regulated, 264 up-regulated) were identified. Among these, several DEGs and DEPs were further verified using quantitative RT-PCR and western blot. Following gene ontology (GO) annotation and KEGG enrichment analysis, we determined that these DEGs and DEPs were significantly enriched in multiple important cellular signaling pathways including NOD-like receptor, Toll-like receptor, TNF, NF-κB, MAPK, cAMP, lysosome, protein processing in endoplasmic reticulum, lipid metabolism, and apoptosis signaling pathways. Significantly, the down-regulated DEGs and DEPs were predominantly associated with apoptosis-regulated elements, inflammatory factors, and antiviral elements that were involved in innate immunity, thus, indicating that BVDV could inhibit apoptosis and the expression of host antiviral genes to facilitate viral replication. Meanwhile, up-regulated DEGs and DEPs were primarily involved in metabolism and autophagy signaling pathways, indicating that BVDV could utilize the host metabolic resources and cell autophagy to promote replication. However, the potential mechanisms BVDV-host interactions required further experimental validation. Our data provide an overview of changes in transcriptomics and proteomics profiles of BVDV-infected MDBK cells, thus, providing an important basis for further exploring the mechanisms of BVDV-host interactions.
Bovine viral diarrhoea virus (BVDV) is the etiologic agent of bovine viral diarrhea-mucosal disease, one of the most important viral diseases in cattle, with inflammatory diarrhea, enteritis, and mucosa necrosis as the major clinical manifestations. NF-κB is an important transcription complex that regulates the expression of genes involved in inflammation and immune responses. NLRP3 inflammasome plays a key role in the development of inflammatory diseases. However, whether the activation of NF-κB is crucial for BVDV infection-induced inflammatory responses remains unclear. The results of our present study showed that BVDV infection significantly activated the NF-κB pathway and promoted the expression of NLRP3 inflammasome components (NLRP3, ASC, pro-caspase 1) as well inflammatory cytokine pro-IL-1β in BVDV-infected bovine cells, resulting in the cleavage of pro-caspase 1 and pro-IL-1β into active form caspase 1 and IL-1β. However, the levels of the NLRP3 inflammasome components and inflammatory cytokines were obviously inhibited, as well the cleavage of pro-caspase 1 and pro-IL-1β in the pre-treated bovine cells with NF-κB-specific inhibitors after BVDV infection. Further, cytopathic biotype BVDV (cpBVDV) Erns and NS5A proteins with their key functional domains contributed to BVDV-induced inflammatory responses via activating the NF-κB pathway were confirmed experimentally. Especially, the NS5A can promote cholesterol synthesis and accelerate its augmentation, further activating the NF-κB signalling pathway. Conclusively, our data elucidate that the activation of NF-κB signaling pathway plays a crucial role in cpBVDV infection-induced inflammatory responses.
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