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OxLDL/?2GPI/anti??2GPI Ab complex induces inflammatory activation via the TLR4/NF??B pathway in HUVECs.

ABSTRACT: Patients with antiphospholipid syndrome have been identified to have higher incidence rates of atherosclerosis (AS) due to the elevated levels of anti??2?glycoprotein I (?2GPI) antibody (Ab). Our previous studies revealed that the anti??2GPI Ab formed a stable oxidized low?density lipoprotein (oxLDL)/?2GPI/anti??2GPI Ab complex, which accelerated AS development by promoting the accumulation of lipids in macrophages and vascular smooth muscle cell. However, the effects of the complex on endothelial cells, which drive the initiation and development of AS, remain unknown. Thus, the present study aimed to determine the proinflammatory roles of the oxLDL/?2GPI/anti??2GPI Ab complex in human umbilical vein endothelial cells (HUVECs) in an attempt to determine the underlying mechanism. Reverse transcription?quantitative PCR, enzymy?linked immunosorbent assay, western blotting and immunofluorescence staining were performed to detect the expressions of inflammation related factors and adhesion molecules. Monocyte?binding assay was used to investigate the effects of oxLDL/?2GPI/anti??2GPI Ab complex on monocyte adhesion to endothelial cells. The results demonstrated that the oxLDL/?2GPI/anti??2GPI Ab complex upregulated the expression of Toll?like receptor (TLR)4 and the levels of NF??B phosphorylation in HUVECs, and subsequently enhanced the expression levels of inflammatory cytokines, including TNF??, IL?1? and IL?6, as well as those of adhesion molecules, such as intercellular adhesion molecule 1 and vascular adhesion molecule 1. In addition, the complex facilitated the recruitment of monocytes by promoting the secretion of monocyte chemotactic protein 1 in HUVECs. Notably, the described effects of the oxLDL/?2GPI/anti??2GPI Ab complex in HUVECs were abolished by either TLR4 or NF??B blockade. In conclusion, these findings suggested that the oxLDL/?2GPI/anti??2GPI Ab complex may induce a hyper?inflammatory state in endothelial cells by promoting the secretion of proinflammatory cytokines and monocyte recruitment, which was discovered to be largely dependent on the TLR4/NK??B signaling pathway.

SUBMITTER: Zhang G

PROVIDER: S-EPMC7789093 | biostudies-literature | 2021 Feb

REPOSITORIES: biostudies-literature

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OxLDL/β2GPI/anti‑β2GPI Ab complex induces inflammatory activation via the TLR4/NF‑κB pathway in HUVECs.

Zhang Guiting G Cai Qianqian Q Zhou Hong H He Chao C Chen Yudan Y Zhang Peng P Wang Ting T Xu Liangjie L Yan Jinchuan J

Molecular medicine reports 20201223 2

Patients with antiphospholipid syndrome have been identified to have higher incidence rates of atherosclerosis (AS) due to the elevated levels of anti‑β2‑glycoprotein I (β2GPI) antibody (Ab). Our previous studies revealed that the anti‑β2GPI Ab formed a stable oxidized low‑density lipoprotein (oxLDL)/β2GPI/anti‑β2GPI Ab complex, which accelerated AS development by promoting the accumulation of lipids in macrophages and vascular smooth muscle cell. However, the effects of the complex on endotheli ...[more]

PMID: 33355374

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Project description:Endothelial cell dysfunction plays a fundamental role in the pathogenesis of atherosclerosis (AS), and endothelial autophagy has protective effects on the development of AS. Our previous study had shown that oxidized low-density lipoprotein/β2-glycoprotein I/anti-β2-glycoprotein I antibody (oxLDL/β2GPI/anti-β2GPI) complex could promote the expressions of inflammatory cytokines and enhance the adhesion of leukocytes to endothelial cells. In the present study, we aimed to assess the effects of oxLDL/β2GPI/anti-β2GPI complex on endothelial autophagy and explore the associated potential mechanisms. Human umbilical vein endothelial cells (HUVECs) and mouse brain endothelial cell line (bEnd.3) were used as models of the vascular endothelial cells. Autophagy was evaluated by examining the expressions of autophagic proteins using western blotting analysis, autophagosome accumulation using transmission electron microscopy, and RFP-GFP-LC3 adenoviral transfection and autophagic flux using lysosome inhibitor chloroquine. The expressions of phospho-PI3K, phospho-AKT, phospho-mTOR, and phospho-eNOS were determined by western blotting analysis. 3-Methyladenine (3-MA) and rapamycin were used to determine the role of autophagy in oxLDL/β2GPI/anti-β2GPI complex-induced endothelial cell dysfunction. We showed that oxLDL/β2GPI/anti-β2GPI complex suppressed the autophagy, evidenced by an increase in p62 protein, a decrease in LC3-II and Beclin1, and a reduction of autophagosome generation in endothelial cells. Moreover, inhibition of autophagy was associated with PI3K/AKT/mTOR and eNOS signaling pathways. Rapamycin attenuated oxLDL/β2GPI/anti-β2GPI complex-induced endothelial inflammation, oxidative stress, and apoptosis, whereas 3-MA alone induced the endothelial injury. Our results suggested that oxLDL/β2GPI/anti-β2GPI complex inhibited endothelial autophagy via PI3K/AKT/mTOR and eNOS signaling pathways and further contributed to endothelial cell dysfunction. Collectively, our findings provided a novel mechanism for vascular endothelial injury in AS patients with an antiphospholipid syndrome (APS) background.

Project description:BACKGROUND:Neuroinflammation is the response of the central nervous system to events that interfere with tissue homeostasis and represents a common denominator in virtually all neurological diseases. Activation of microglia, the principal immune effector cells of the brain, contributes to neuronal injury by release of neurotoxic products. Toll-like receptor 4 (TLR4), expressed on the surface of microglia, plays an important role in mediating lipopolysaccharide (LPS)-induced microglia activation and inflammatory responses. We have previously shown that curcumin and some of its analogues harboring an ?,?-unsaturated 1,3-diketone moiety, able to coordinate the magnesium ion, can interfere with LPS-mediated TLR4-myeloid differentiation protein-2 (MD-2) signaling. Fluoroquinolone (FQ) antibiotics are compounds that contain a keto-carbonyl group that binds divalent ions, including magnesium. In addition to their antimicrobial activity, FQs are endowed with immunomodulatory properties, but the mechanism underlying their anti-inflammatory activity remains to be defined. The aim of the current study was to elucidate the molecular mechanism of these compounds in the TLR4/NF-?B inflammatory signaling pathway. METHODS:The putative binding mode of five FQs [ciprofloxacin (CPFX), levofloxacin (LVFX), moxifloxacin, ofloxacin, and delafloxacin] to TLR4-MD-2 was determined using molecular docking simulations. The effect of CPFX and LVFX on LPS-induced release of IL-1? and TNF-? and NF-?B activation was investigated in primary microglia by ELISA and fluorescence staining. The interaction of CPFX and LVFX with TLR4-MD-2 complex was assessed by immunoprecipitation followed by Western blotting using Ba/F3 cells. RESULTS:CPFX and LVFX bound to the hydrophobic region of the MD-2 pocket and inhibited LPS-induced secretion of pro-inflammatory cytokines and activation of NF-?B in primary microglia. Furthermore, these FQs diminished the binding of LPS to TLR4-MD-2 complex and decreased the resulting TLR4-MD-2 dimerization in Ba/F3 cells. CONCLUSIONS:These results provide new insight into the mechanism of the anti-inflammatory activity of CPFX and LVFX, which involves, at least in part, the activation of TLR4/NF-?B signaling pathway. Our findings might facilitate the development of new molecules directed at the TLR4-MD-2 complex, a potential key target for controlling neuroinflammation.

Dataset Information

OxLDL/?2GPI/anti??2GPI Ab complex induces inflammatory activation via the TLR4/NF??B pathway in HUVECs.

Publications

OxLDL/β2GPI/anti‑β2GPI Ab complex induces inflammatory activation via the TLR4/NF‑κB pathway in HUVECs.

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