Extracellular Matrix Disorganization Caused by ADAMTS16 Deficiency Leads to Bicuspid Aortic Valve With Raphe Formation.

BACKGROUND: A better understanding of the molecular mechanism of aortic valve development and bicuspid aortic valve (BAV) formation would significantly improve and optimize the therapeutic strategy for BAV treatment. Over the past decade, the genes involved in aortic valve development and BAV formation have been increasingly recognized. On the other hand, ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) gene family members have been reported to be able to modulate cardiovascular development and diseases. The present study aimed to further investigate the roles of ADAMTS family members in aortic valve development and BAV formation. METHODS: Morpholino-based ADAMTS family gene-targeted screening for zebrafish heart outflow tract phenotypes combined with DNA sequencing in a 304 cohort BAV patient registry study was initially carried out to identify potentially related genes. Both ADAMTS gene-specific fluorescence in situ hybridization assay and genetic tracing experiments were performed to evaluate the expression pattern in the aortic valve. Accordingly, related genetic mouse models (both knockout and knockin) were generated using the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9) method to further study the roles of ADAMTS family genes. The lineage-tracing technique was used again to evaluate how the cellular activity of specific progenitor cells was regulated by ADAMTS genes. Bulk RNA sequencing was used to investigate the signaling pathways involved. Inducible pluripotent stem cells derived from both BAV patients and genetic mouse tissue were used to study the molecular mechanism of ADAMTS. Immunohistochemistry was performed to examine the phenotype of cardiac valve anomalies, especially in the extracellular matrix components. RESULTS: ADAMTS genes targeting and phenotype screening in zebrafish and targeted DNA sequencing on a cohort of patients with BAV identified ADAMTS16 (a disintegrin and metalloproteinase with thrombospondin motifs 16) as a BAV-causing gene and found the ADAMTS16 p. H357Q variant in an inherited BAV family. Both in situ hybridization and genetic tracing studies described a unique spatiotemporal pattern of ADAMTS16 expression during aortic valve development. Adamts16+/- and Adamts16+/H355Q mouse models both exhibited a right coronary cusp-noncoronary cusp fusion-type BAV phenotype, with progressive aortic valve thickening associated with raphe formation (fusion of the commissure). Further, ADAMTS16 deficiency in Tie2 lineage cells recapitulated the BAV phenotype. This was confirmed in lineage-tracing mouse models in which Adamts16 deficiency affected endothelial and second heart field cells, not the neural crest cells. Accordingly, the changes were mainly detected in the noncoronary and right coronary leaflets. Bulk RNA sequencing using inducible pluripotent stem cells-derived endothelial cells and genetic mouse embryonic heart tissue unveiled enhanced FAK (focal adhesion kinase) signaling, which was accompanied by elevated fibronectin levels. Both in vitro inducible pluripotent stem cells-derived endothelial cells culture and ex vivo embryonic outflow tract explant studies validated the altered FAK signaling. CONCLUSIONS: Our present study identified a novel BAV-causing ADAMTS16 p. H357Q variant. ADAMTS16 deficiency led to BAV formation.

[Research progress on the effect of mitochondrial network remodeling on macrophages].

Remodeling of the mitochondrial network is an important process in the maintenance of cellular homeostasis and is closely related to mitochondrial function. Interactions between the biogenesis of new mitochondria and the clearance of damaged mitochondria (mitophagy) is an important manifestation of mitochondrial network remodeling. Mitochondrial fission and fusion act as a bridge between biogenesis and mitophagy. In recent years, the importance of these processes has been described in a variety of tissues and cell types and under a variety of conditions. For example, robust remodeling of the mitochondrial network has been reported during the polarization and effector function of macrophages. Previous studies have also revealed the important role of mitochondrial morphological structure and metabolic changes in regulating the function of macrophages. Therefore, the processes that regulate remodeling of the mitochondrial network also play a crucial role in the immune response of macrophages. In this paper, we focus on the molecular mechanisms of mitochondrial regeneration, fission, fusion, and mitophagy in the process of mitochondrial network remodeling, and integrate these mechanisms to investigate their biological roles in macrophage polarization, inflammasome activation, and efferocytosis.

Feature Sequencing Method of Industrial Control Data Set Based on Multidimensional Evaluation Parameters.

The industrial control data set has many features and large redundancy, which has a certain impact on the training speed and classification results of the neural network anomaly detection algorithm. However, features are independent of each other, and dimension reduction often increases the false positive rate and false negative rate. The feature sequencing algorithm can reduce this effect. In order to select the appropriate feature sequencing algorithm for different data sets, this paper proposes an adaptive feature sequencing method based on data set evaluation index parameters. Firstly, the evaluation index system is constructed by the basic information of the data set, the mathematical characteristics of the data set, and the association degree of the data set. Then, the selection model is obtained by the decision tree training with the data label and the evaluation index, and the suitable feature sequencing algorithm is selected. Experiments were conducted on 11 data sets, including Batadal data set, CICIDS 2017, and Mississippi data set. The sequenced data sets are classified by ResNet. The accuracy of the sequenced data sets increases by 2.568% on average in 30 generations, and the average time reduction per epoch is 24.143%. Experiments show that this method can effectively select the feature sequencing algorithm with the best comprehensive performance.

Detection of miR-122 by fluorescence real-time PCR in blood from patients with chronic hepatitis B and C infections.

BACKGROUND: This study aims to determine whether relative miR-122 levels in peripheral blood are correlated with chronic hepatitis B (CHB) and chronic hepatitis C (CHC) virus infection and viral replication to determine whether miR-122 can be a new marker for liver injury. METHODS: MicroRNA (miRNA) was extracted from the peripheral blood of 20 CHB patients, 20 CHC patients, and 20 healthy controls. The levels of miR-122 were determined using fluorescence real-time reverse transcription PCR. Then, the associations of miR-122 with CHB and CHC were analyzed, and its correlation with other markers of liver function and viral replication were determined. RESULTS: The expression level of miR-122 in patients with CHB was significantly higher when compared to subjects in the control group (P = 0.007) or CHC patients (P = 0.005). Furthermore, the miR-122 level in patients with CHC was somewhat higher when compared to healthy controls (66% higher), but the difference was not statistically significant (P = 0.229). MiR-122 levels were significantly correlated with ALT (correlation coefficient [R] = 0.7, P < 0.001), AST (R = 0.71, P < 0.001), and HBV NA (R = 0.9, P < 0.001). The regression analysis indicated that the AUC of miR-122 levels in the diagnosis of CHB was 0.87, with a sensitivity of 0.8 and a specificity of 0.8. CONCLUSION: MiR-122 can be used to distinguish healthy persons and patients with CHB infection with high sensitivity and specificity. These present findings presented that the complex and context-specific associations of miR-122 with liver diseases, suggesting that this may be a promising marker for liver injury.

Role of TLR­4 in anti­ß2­glycoprotein I­induced activation of peritoneal macrophages and vascular endothelial cells in mice.

Anti­phospholipid syndrome (APS) is a systematic autoimmune disease that is associated with presence of antiphospholipid antibodies (aPL), recurrent thrombosis, and fetal morbidity in pregnancy. Toll­like receptor­4 (TLR­4), a member of TLR family, is known to have a fundamental role in pathogen recognition and activation of innate immunity. The ß2­glycoprotein I (ß2GPI), a protein circulating in the blood at a high concentration, is able of scavenging lipopolysaccharide (LPS) and clear unwanted anionic cellular remnants, such as microparticles, from the circulation. Our previous study demonstrated that TLR­4 and its signaling pathways contribute to the upregulation of pro­coagulant factors and pro­inflammatory cytokines in monocytes induced by anti­ß2GPI in vitro. The present study aimed to define the roles of TLR­4 in vivo. C3H/HeN mice (TLR­4 intact) and C3H/HeJ mice (TLR­4 defective) were stimulated with an intraperitoneal injection with anti­ß2GPI­immunoglobulin G(IgG), then peritoneal macrophages and vascular endothelial cells (VECs) were extracted from treated mice, and analyses were conducted on the expression profiles of pro­inflammatory cytokines and adhesion molecules. The results demonstrated that the expression of pro­inflammatory cytokines, including tumor necrosis factor­α (TNF­α), interleukin (IL)­1ß and IL­6, in the peritoneal macrophages, and adhesion molecules, including intercellular cell adhesion molecule­1 (ICAM­1), vascular cell adhesion molecule­1 (VCAM­1) and E­selectin, in VECs of C3H/HeN mice (TLR­4 intact) were significantly higher than those of C3H/HeJ mice (TLR­4 defective). The phosphorylation levels of p38 mitogen­activated protein kinase (MAPK) and nuclear factor­κB (NF­κB) p65 in peritoneal macrophages and VECs from C3H/HeN mice stimulated with anti­ß2GPI­IgG were significantly increased compared with those from C3H/HeJ mice (TLR­4 defective). The isotype control antibody (NR­IgG) had no such effects on peritoneal macrophages and VECs. Furthermore, the inhibitors of TLR­4, p38 MAPK and NF­κB may significantly reduce the anti­ß2GPI­IgG­induced TNF­α, IL­1ß and IL­6 mRNAs expression in the peritoneal macrophages from TLR­4 intact mice. The results indicated that a TLR­4 signal transduction pathway is involved in anti­ß2GPI­IgG­induced activation of peritoneal macrophages and VECs. This study has provided a basis for subsequent investigations to elucidate the pathological mechanisms underlying anti­phospholipid syndrome.

USP4 positively regulates RLR-induced NF-κB activation by targeting TRAF6 for K48-linked deubiquitination and inhibits enterovirus 71 replication.

Retinoic acid-inducible gene I-like receptor (RLR) is one of the most important pattern recognition receptors of the innate immune system that detects positive and/or negative stranded RNA viruses. Subsequently, it stimulates downstream transcription of interferon regulatory factor 3 (IRF3) and nuclear factor κB (NF-κB) inducing the production of interferons (IFNs) and inflammatory cytokines. Tumour necrosis factor receptor associated factor 6 (TRAF6) is a key protein involved in the RLR-mediated antiviral signalling pathway, recruiting additional proteins to form a multiprotein complex capable of activating the NF-κB inflammatory pathway. Despite TRAF6 playing an important role in regulating host immunity and viral infection, the deubiquitination of TRAF6 induced by viral infection remains elusive. In this study, we found that enterovirus 71 (EV71) infection attenuated the expression of Ubiquitin-specific protease 4 (USP4) in vitro and in vivo, while overexpression of USP4 significantly suppressed EV71 replication. Furthermore, it was found that EV71 infection reduced the RLR signalling pathway and enhanced the degradation of TRAF6. USP4 was also found to interact with TRAF6 and positively regulate the RLR-induced NF-κB signalling pathway, inhibiting the replication of EV71. Therefore, as a novel positive regulator of TRAF6, USP4 plays an essential role in EV71 infection by deubiquitinating K48-linked ubiquitin chains.

[Vasodilation effect and mechanism of extraction of Tongmai Yangxin Pills (TMYX) on isolated rat mesenteric artery].

The aim of the present study is to evaluate the vasodilation effects of Tongmai Yangxin Pills (TMYX) on rat mesenteric artery as well as its mechanism of action. The relaxation effects of TMYX extracts with different concentrations were determined on isolated rat mesenteric artery in normal condition as well as pretreating by phenylephrine and KCl. Vascular relaxation effects of TMTX were also determined in mesenteric artery preincubated with L-ANME and indomethacin or in endothelium denuded mesenteric artery. Moreover, effects of TMYX by 50 mg·L⁻¹ on NO secretion and the phosphorylation of eNOS in a cellular model of human umbilical vein endothelial cell (HUVEC) pretreated with or without L-NAME were also observed. The experimental results showed that TMYX has no obvious effect on vasodilation of arteries in normal or KCl pretreated condition, while it can dose-dependently relax the rat mesenteric artery with intact endothelium stimulated with phenylephrine at a maximal diastolic rate of (64.71±10.03)%. After preincubating with L-NAME for 15 min or removal of mesenteric artery endothelium, the maximal diastolic rate was decreased to (35.77±8.93)% and (25.85±10.84)% respectively. However, preincubating with indomethacin had no inhibitory effect on TMYX induced vascular relaxation. Meanwhile, TMYX at 50 mg·L⁻¹ could increase the expression of P-eNOS and the secretion of NO in HUVEC. L-NAME significantly inhibited NO release and phosphorylation of eNOS induced by TMYX. The results suggested TMYX exerted endothelium-dependent relaxation effects against PE-induced contractions of isolated rat mesenteric artery through NO-cGMP signaling pathway.

Reduced neurotrophic factor level is the early event before the functional neuronal deficiency in high-fat diet induced obese mice.

Neurodegeneration is considered one of the possible complications of high fat diet (HFD) induced obesity. Much evidence has shown the close relationship between HFD and dementia at comparatively later stage of neuronal injury. It is so far not clear that the initial events of neuronal injury resulting from HFD and obesity. In the present research, obese mouse model achieved by 3-month HFD was applied for the investigation of the possible neuronal deficiency before the obvious cognitive decline. We found that 3-month HFD has already increased the average level of body weight of mice. But almost no obvious cognitive defect was observed. At such time point, we detected the cleavage of amyloid precursor protein (APP), including the expression and maturation level of α- and ß-secretase and proteolytic fragment soluble APP. Results showed similar readout between HFD and normal diet (ND) mice. Besides, neuronal inflammation and brain-blood barrier permeability were also detected. No obvious changes could be observed between HFD and ND mice. Surprisingly, the first detectable neuronal changes was showed to be the downregulation of some neurotrpic factors, like neuronal growth factor ß and brain derived neurotrophic factor, together with the activity of specific receptors, like Trk receptor phosphorylation. All the data piled up indicated that the early neuronal change in HFD induced obese mice was the downregulation of some neurotrophic factors. The results may provide the potential clue to therapeutic and preventive strategy for HFD induced cognitive decline.

[TLR2 blockade reduces TNF-α expression induced by ß2GP1/anti-ß2GP1 complex in mouse peritoneal macrophages].

OBJECTIVE: To investigate the role of Toll-like receptor 2 (TLR2) in ß2-glycoprotein 1/anti-ß2-glycoprotein 1 (ß2GP1/anti-ß2GP1)-mediated tumor necrosis factor α (TNF-α) expression in mouse peritoneal macrophages. METHODS: The peritoneal macrophages from BALB/c mice were treated with ß2GP1/anti-ß2GP1 complex, agonist of TLR2 (Pam3CSK4) and agonist of TLR4 [lipopolysaccharide (LPS)], inhibitor of TLR2 [monoclonal IgG to mouse TLR2 (anti-mTLR2-IgG)] and inhibitor of TLR4 (TAK-242) in vitro. The mRNA level of TNF-α in the peritoneal macrophages was tested by real-time quantitative PCR, the protein expression of TNF-α was detected by Western blotting and immunofluorescence cytochemistry, and the expression of TLR2 on the surface of the peritoneal macrophages was assessed by flow cytometry. RESULTS: The mRNA and protein expression of TNF-α was significantly enhanced in mouse peritoneal macrophages treated with ß2GP1/anti-ß2GP1 complex, Pam3CSK4 and LPS. Anti-mTLR2-IgG could inhibit the effects of the above stimuli on TNF-α expression, but its effects were weaker than those of TAK-242. Meanwhile, the combination of anti-mTLR2-IgG and TAK-242 did not show much stronger inhibitory effects. Flow cytometry analysis showed that expression of TLR2 was enhanced by ß2GP1/anti-ß2GP1 complex, Pam3CSK4 and LPS in mouse peritoneal macrophages. However, anti-mTLR2-IgG, or TAK-242, or combination of both could not inhibit TLR2 expression in macrophages. CONCLUSION: Both TLR4 and TLR2 could increase the stimulating effect of ß2GP1/anti-ß2GP1 complex on the expression of TNF-α in mouse peritoneal macrophages.

The effect of Toll-like receptor 4 on ß2-glycoprotein I-induced B cell activation in mouse model.

Our previous study demonstrated that Toll-like receptor 4 (TLR4) plays a vital role in the maturation of bone marrow-derived dendritic cells (BMDCs) from the mice immunized with human ß2-glycoprotein I (ß2GPI). However, the roles of TLR4 in the activation of B cells and production of anti-ß2GPI antibodies in vivo have been rarely studied. This study aimed to investigate the activation of B cells from TLR4-defective (C3H/HeJ) and TLR4-intact (C3H/HeN) mice pre-immunized with human ß2GPI. After ß2GPI injection, the level of anti-ß2GPI antibody in the serum of TLR4-defective and TLR4-intact mice was gradually increased and the number and size of germinal centers in the spleen were also significantly increased. Compared with C3H/HeJ mice, we observed significantly higher anti-ß2GPI antibody titer and more germinal centers in C3H/HeN mice. Moreover, the ß2GPI-induced expression of CD40L, CD40, CD80, CD86 and MHC II in C3H/HeN mice was significantly higher than that in C3H/HeJ mice. Furthermore, the ß2GPI-induced expression of B cell activating factor (BAFF) in the spleen and IL-6 and IL-10 in B cells from C3H/HeN mice was also significantly increased compared to C3H/HeJ mice. Taken together, our results suggest that TLR4 is required for the activation of B cells and the production of autoantibody in mice treated with ß2GPI, but the immunological mechanisms of antiphospholipid syndrome (APS) need further investigation.

[TLR4 promotes the activation of B cells in ß2GP1-immunized mice].

OBJECTIVE: To investigate the effect of Toll-like receptor 4 (TLR4) on B cell activation in ß2-glycoprotein I (ß2GP1)-immunized mouse models. METHODS: The healthy TLR4+/+ C3H/HeN mice and TLR4-/- C3H/HeJ mice were randomly divided into two groups: ß2GP1 group and normal saline (NS) group. The mice were immunized with human ß2GP1 or equal amount of NS. The primary immunization was done by subcutaneous multi-point injection. The booster immunization was performed by intraperitoneal injection and the last immunization by intravenous injection. The titer of anti-ß2GP1 antibody in mouse sera was evaluated by indirect ELISA. HE staining was used to observe the changes of mouse germinal centers. The expression of CD40 ligand (CD40L) in the germinal centers was detected by immunohistochemistry. And the levels of co-stimulatory molecules CD80 and CD86 in the spleen were determined by flow cytometry. RESULTS: The high titer of anti-ß2GP1 antibody could be detected in mouse sera following immunization with ß2GP1. And the titer of the antibody in TLR4+/+ C3H/HeN mice was higher than that in TLR4-/- C3H/HeJ mice. Compared with NS group, the CD40L, CD19+CD80+ cells and CD19+CD86+ cells in C3H/HeN and C3H/HeJ mice were significantly up-regulated by the treatment of ß2GPI, and the cell numbers in the C3H/HeJ mice were lower than those in the C3H/HeN mice. The germinal centers in ß2GP1-immunized mice were bigger and more than those in NS group, and the number of the germinal centers in TLR4+/+ C3H/HeN mice was more than that in TLR4-/- C3H/HeJ mice. CONCLUSION: TLR4 plays an important role in the process of B cell activation in ß2GP1-immunized mouse models.

TLR4 is involved in the pathogenic effects observed in a murine model of antiphospholipid syndrome.

Antiphospholipid (aPL)/anti-ß2-glycoprotein I (ß2GPI) antibodies are considered to play a pivotal pathogenic role in antiphospholipid syndrome (APS) by inducing an intracellular signaling and procoagulant/proinflammatory phenotype that leads to thrombosis. There is increasing evidence that Toll-like receptor 4 (TLR4) could serve as an important molecule for anti-ß2GPI recognition on target cells. However, few studies have focused on the effects of TLR4 in in vivo models. Here, we investigated the role of TLR4 in the pathogenic effects of aPL/anti-ß2GPI more precisely using TLR4-intact (C3H/HeN) and TLR4-defective (C3H/HeJ) mice. C3H/HeN and C3H/HeJ mice were injected with either IgG isolated from patient with APS (IgG-APS) or epitope-specific anti-ß2GPI purified from ß2GPI peptide-immunized rabbits. We found that, following anti-ß2GPI injections and vascular injury, thrombus formation in both the carotid artery and femoral vein was markedly reduced in C3H/HeJ mice when compared with C3H/HeN mice. IgG-APS or anti-ß2GPI-induced carotid artery and peritoneal macrophage tissue factor activity/expression was significantly lesser in C3H/HeJ than in C3H/HeN mice. Furthermore, the IgG-APS or anti-ß2GPI induced expression of VCAM-1, ICAM-1, and E-selectin in the aorta and of IL-1ß, IL-6, and TNF-α in peritoneal macrophages of C3H/HeJ mice was also significantly reduced compared to C3H/HeN mice. Together, these data suggest that TLR4 is involved in the pathogenic effects of aPL/anti-ß2GPI antibodies in vivo.

[Effect of ß2GPI immunization on mouse T helper cell subset differentiation].

OBJECTIVE: To explore the mechanism of beta2 glycoprotein I (ß2GPI)-caused T helper cell subset differentiation and anti-ß2GPI antibody (aß2GPI Ab) production in mice. METHODS: The healthy BALB/c mice were randomly divided into two groups: normal saline (NS) group and ß2GPI group. The mice were immunized i.v. with either human ß2GPI or equal amount of NS for a total of two or four times at 2-week intervals. The titer of aß2GPI Ab in mice serum was evaluated by indirect ELISA. The expressions of GATA binding protein 3 (GATA3), interleukin 4 (IL-4), T-box expressed in T cells (T-bet), interferon-γ (IFN-γ) and Foxp3 mRNAs in mouse splenic cells were detected by real-time quantitative PCR (qRT-PCR), and the percentage of GATA3+ or Foxp3+ cells in lymphocytes of mouse spleen was detected by flow cytometry. RESULTS: The high titer (1:100,000) of aß2GPI Ab was detected in mouse serum following the two times of immunization with ß2GPI. The expressions of Th2 specific markers GATA3 and IL-4 mRNAs and the percentage of GATA3+ cells in lymphocytes were higher than those of NS treated group. After four times of immunization with ß2GPI, the titer of aß2GPI Ab in mouse serum was elevated to a higher level (>1:100,000). The expressions of GATA3, Th1 specific markers T-bet and IFN-γ, and regulatory T cell (Treg) specific marker Foxp3 mRNAs in mouse spleen cells, but no significant change was found in IL-4 mRNA expression. Furthermore, the percentage of GATA3+ cells in mouse spleen increased, while the percentage of Foxp3+ cells decreased. CONCLUSION: ß2GPI immunization could induce Th2 cell bias polarization and Th1 cell and Treg suppression in mice, which might contribute to the production of aß2GPI Ab.

[Preparation and application of a polyclonal antibody against the peptides of ß2glycoprotein 1].

OBJECTIVE: To prepare a polyclonal antibody against human and murine ß2 glycoprotein 1 (ß2GP1) antigen with chemically synthesized ß2GP1 peptides, and identify its specificity and pathogenicity. METHODS: The peptides from the NH2-terminal 35th-51th amino acids of ß2GP1 were synthesized by standard Fmoc assay, and then used to immunize New Zealand white rabbits after coupling with keyhole limpet hemocyanin (KLH). The polyclonal antibody in the rabbit sera was purified by protein G column. The titer and specificity of the polyclonal antibody were determined by ELISA and Western blot analysis. The total RNA was extracted and the protein lysates were collected from C3H/HeN mouse peritoneal macrophages treated with the above anti-ß2GP1 peptides antibody/ß2GP1 complexes in vitro. And the tissue factor (TF) mRNA and protein expression in the peritoneal macrophages were detected by real-time quantitative PCR and Western blotting. The activation of p38 and NF-κB p65 induced by anti-ß2GP1 peptides antibody/ß2GP1 complexes was determined by Western blotting using phosphor-specific antibodies. Experimental antiphospholipid antibody syndrome (EAPS) mouse model was established in C3H/HeN mice by intraperitoneal injection of anti-ß2GP1 peptides antibody in vivo. The titers of anti-ß2GP1 antibodies in the mouse peripheral blood and the activated partial thromboplastin time (APTT) were detected. RESULTS: The purity of chemically synthesized ß2GP1 peptides was 94%, which met the immunogen standard. The titer of antiserum of the rabbit immunized with ß2GP1 peptide coupling with KLH was over 1:32 000. Western blotting showed that the anti-ß2GP1 peptides antibody could specifically recognize both human and mouse ß2GP1. Furthermore, ELISA showed that the antibody could specifically bind to ß2GP1 cryptic epitope. In vitro experiments demonstrated that the anti-ß2GP1 peptides antibody/ß2GP1 co mplexes could enhance p38 and NF-κB p65 phosphorylation in mouse peritoneal macrophages and induce TF mRNA and protein expression. Moreover, EAPS mouse model induced by anti-ß2GP1 peptide antibody was successfully established, in which the titers of anti-ß2GP1 antibody in mouse peripheral blood were greater than 1:3200 and APTT was significantly shorter that that of control group. CONCLUSION: The anti-ß2GP1 peptide antibody we prepared could specifically recognize both human and mouse ß2GP1 and specifically bind to ß2GP1 cryptic epitope. It was also proved to have the pathogenic effect.

oxLDL/ß2GPI/anti-ß2GPI complex induced macrophage differentiation to foam cell involving TLR4/NF-kappa B signal transduction pathway.

Macrophage-derived foam cell formation is a hallmark of atherosclerosis. It has been reported that oxidized low density lipoprotein (oxLDL) inducing formation of foam cells and expression of inflammatory molecules are partly mediated by toll-like receptor 4 (TLR4)/nuclear factor kappa B (NF-κB) pathway. However, whether oxLDL/ß2-glycoprotein I/anti-ß2-glycoprotein I (oxLDL/ß2GPI/anti-ß2GPI) complex enhanced formation of foam cells involving TLR4/NF-κB pathway or not has never been explored. In the current study, we focused on investigating the transformation of peritoneal macrophages from BALB/c mice into foam cells induced by the three complexes, and the involvement of TLR4 as well as its downstream signal molecule NF-κB. The results showed that treatment of macrophages with oxLDL/ß2GPI/anti-ß2GPI complex could markedly increase intracellular lipid loading and expression of TLR4, phosphorylated NF-κB p65 (p-NF-κB p65), monocyte chemoattractant protein-1 (MCP-1), as well as tissue factor (TF). The oxLDL and oxLDL/ß2GPI/anti-ß2GPI complex induced formation of foam cells and expression of p-NF-κB p65 were significantly reduced, while macrophages were pre-treated with TLR4 inhibitor TAK-242. Meanwhile, both TAK-242 and NF-κB inhibitor PDTC could remarkably inhibit oxLDL, oxLDL/ß2GPI/anti-ß2GPI complex, as well as LPS increased MCP-1 and TF levels. Nevertheless, ß2GPI/anti-ß2GPI complex-induced MCP-1 and TF mRNA expression were inhibited by TAK-242 rather than PDTC, although TF activity was significantly reduced by both of the inhibitors. In conclusion, our results indicate that oxLDL/ß2GPI/anti-ß2GPI complex could enhance the conversion of macrophages into foam cells and the process may be at least partly mediated by TLR4/NF-κB pathway, which may contribute to the accelerated development of atherosclerosis in APS.

[Toll-like receptor 4 promotes macrophage foam cell formation induced by oxidized low-density/ß2-glycoprotein I/ß2-glycoprotein I antibodies complex].

OBJECTIVE: To explore the role of toll-like receptor 4 (TLR4) on oxidized low-density/ß2-glycoprotein I/ß2-glycoprotein I (ox-LDL/ß2GPI/anti-ß2GPI) antibodies complex induced macrophage foam cell formation. METHODS: The peritoneal macrophages were separated from TLR4 intact C3H/HeN mice and TLR4 defective C3H/HeJ mice. The cells were treated with ox-LDL, ox-LDL/ß2GPI, ox-LDL/anti-ß2GPI, anti-ß2GPI/ß2GPI, ox-LDL/ß2GPI/anti-ß2GPI, lipopolysaccharide (LPS) for 48 h and the foam cells were identified by Oil red O staining for intracellular lipids. The total cellular RNA and the protein lysates were collected. The levels of tissue factor (TF) mRNA in two groups were detected by real-time PCR (RT-PCR), and the expression of phosphorylated nuclear factor-κB (NF-κB) p65 was detected by Western blotting. Monocyte chemotactic protein-1 (MCP-1) secretion from peritoneal macrophages was determined by enzyme linked immunosorbent assay (ELISA) kits. RESULTS: Compared with C3H/HeJ mice, lipid droplets in the cytoplasm of peritoneal macrophages from C3H/HeN mice were significantly increased and phosphorylation-NF-κB expression was significantly upregulated after stimulating by ox-LDL/ß2GPI/anti-ß2GPI complex (P < 0.01). TF mRNA and MCP-1 expression were also upregulated post ox-LDL/ß2GPI/anti-ß2GPI complex stimulation [TF mRNA: 0.041 ± 0.023 vs. 0.005 ± 0.003; MCP-1: (6 200.2 ± 6.4) pg/ml vs. (803.3 ± 5.5) pg/ml, P < 0.01]. CONCLUSION: TLR4 can enhance ox-LDL/ß2GPI/anti-ß2GPI complex induced peritoneal macrophage foam cell formation via upregulating phosphorylation-NF-κB, TF and MCP-1 expression.

[Role of Toll-like receptor 4 in mouse bone marrow-derived dendritic cell maturation induced by ß2GPI].

OBJECTIVE: To investigate the role of Toll-like receptor 4 (TLR4) in mouse bone marrow-derived dendritic cell maturation induced by beta-2-glycoprotein I (ß2GPI). METHODS: The recombinant murine granulocyte-macrophage colony-stimulating factor (rmGM-CSF) and interleukin-4 (rmIL-4) were utilized to induce bone marrow cells from TLR4 intact (C3H/HeN) mice and TLR4 defective (C3H/HeJ) mice into immature dendritic cells (iDCs) in vitro. The iDCs were further stimulated by ß2GPI or LPS (as a positive control), and the morphological changes of cells were observed under an inverted light microscope, the expression changes of surface molecules on cells were analyzed with flow cytometry, and the levels of IL-12 and TNF-α secreted by cells were detected by ELISA. RESULTS: The DCs from C3H/HeN mice showed more protrusions and displayed more mature in morphology in response to ß2GPI or LPS, compared with those from C3H/HeJ mice. The expressions of surface molecules, CD11c and MHC-II, were higher on DCs from C3H/HeN mice than those from C3H/HeJ mice (P<0.05). And the levels of IL-12 and TNF-α secreted by DCs from C3H/HeN mice were also higher than those from C3H/HeJ mice (P<0.01). CONCLUSION: TLR4 plays an important role in the process of mouse bone marrow-derived dendritic cell maturation induced by ß2GPI.