Effects of apolipoprotein H downregulation on lipid metabolism, fatty liver disease, and gut microbiota dysbiosis.

Apolipoprotein H (APOH) downregulation can cause hepatic steatosis and gut microbiota dysbiosis. However, the mechanism by which APOH-regulated lipid metabolism contributes to metabolic dysfunction-associated steatotic liver disease (MASLD) remains undetermined. Herein, we aim to explore the regulatory effect of APOH, mediated through various pathways, on metabolic homeostasis and MASLD pathogenesis. We analyzed serum marker levels, liver histopathology, and cholesterol metabolism-related gene expression in global ApoH-/- C57BL/6 male mice. We used RNA sequencing and metabolomic techniques to investigate the association between liver metabolism and bacterial composition. Fifty-two differentially expressed genes were identified between ApoH-/- and WT mice. The mRNA levels of de novo lipogenesis genes were highly upregulated in ApoH-/- mice than in WT mice. Fatty acid, glycerophospholipid, sterol lipid, and triglyceride levels were elevated, while hyodeoxycholic acid levels were significantly reduced in the liver tissues of ApoH-/- mice than in those of WT mice. Microbial beta diversity was lower in ApoH-/- mice than in WT mice, and gut microbiota metabolic functions were activated in ApoH-/- mice. Moreover, ApoH transcripts were downregulated in patients with MASLD, and APOH-related differential genes were enriched in lipid metabolism. Open-source transcript-level data from human metabolic dysfunction-associated steatohepatitis livers reinforced a significant association between metabolic dysfunction-associated steatohepatitis and APOH downregulation. In conclusion, our studies demonstrated that APOH downregulation aggravates fatty liver and induces gut microbiota dysbiosis by dysregulating bile acids. Our findings offer a novel perspective on APOH-mediated lipid metabolic dysbiosis and provide a valuable framework for deciphering the role of APOH in fatty liver disease.

Alcohol-dependent downregulation of apolipoprotein H exacerbates fatty liver and gut microbiota dysbiosis in mice.

BACKGROUND: Alcohol-related liver disease (ALD) is a major chronic liver ailment caused by alcohol overconsumption and abuse. Apolipoprotein H (APOH) participates in lipid metabolism and might have a potential regulatory role in ALD. Therefore, this study aimed to explore the effects of ApoH on alcohol-induced liver injury and gut microbiota dysbiosis. METHODS: ApoH-/- mice were generated and the synergic alcoholic steatohepatitis mouse model was constructed, which were used to assess liver function and pathological changes. RESULTS: ApoH-/- mice clearly exhibited spontaneous steatohepatitis. Severe hepatic steatosis was observed in alcohol-fed WT and ApoH-/- mice, in which ApoH expression was reduced post alcohol consumption. Moreover, RNA-seq and KEGG pathway analyses indicated that differential expression genes enriched in lipid metabolism and oxidation-reduction process between in alcohol-fed ApoH-/- mice and pair-fed control mice. Finally, gut microbiota diversity and composition were assessed by 16S rRNA Illumina next-generation sequencing. Alpha diversity of enterobacteria was lower in ApoH-/- mice with ethanol feeding than in ethanol-fed WT mice and all control-fed mice (P < 0.05). Moreover, KEGG enrichment analysis, using PICRUSt software, revealed that metabolic functions were activated in the gut microorganisms of ApoH-/- mice with ethanol feeding (P < 0.05). CONCLUSIONS: Alcohol-downregulated ApoH expression, leading to the progress of fatty liver disease and gut microbiota dysbiosis.

In vivo evidence of angiogenesis inhibition by ß2-glycoprotein I subfractions in the chorioallantoic membrane of chicken embryos.

The vascular network expansion and functioning are important factors affecting normal intra-uterine fetal development. This study addressed the previously reported antiangiogenic potential of beta-2-glycoprotein I (ß2GPI) in vivo in the chick embryo model of angiogenesis. The effects of two naturally occurring ß2GPI forms on the development of the chorioallantoic membrane (CAM) vessels and the chicken embryo were investigated. ß2GPI monomers and dimers were obtained by fractioned purification and characterized using SDS-PAGE, immunoblot, and ELISA. The egg exposure was performed by injection of small volumes of 2.5 µg/mL solutions of the ß2GPI subfractions. Angiogenesis was evaluated through quantitative measurements of vascular architecture parameters in the captured CAM images, using computational analysis of texture contrasts and computer vision techniques. Quantitative information was assigned to the CAM vasculature modifications. In vivo, the ß2GPI dimer completely halted the formation of CAM vessels and led to embryo death after 48 h of exposure. The ß2GPI monomer allowed the embryo to develop up to the 10th day, despite early changes of CAM vessels. The impaired normal vessel growth proceeded as a self-limited effect. The ß2GPI monomer-exposed eggs showed reduced vascularization on the 6th day of incubation, but embryos were viable on the 10th day of incubation, with ingurgitated CAM vessels implying sequelae of the angiogenesis inhibition. Both subfractions impaired CAM vasculature development. The ß2GPI dimer proved to be largely more harmful than the ß2GPI monomer. ß2GPI modification by cleavage or dimerization may play a role in angiogenesis control in vivo.

OxLDL/ß2GPI/anti­ß2GPI Ab complex induces inflammatory activation via the TLR4/NF­κB pathway in HUVECs.

Patients with antiphospholipid syndrome have been identified to have higher incidence rates of atherosclerosis (AS) due to the elevated levels of anti­ß2­glycoprotein I (ß2GPI) antibody (Ab). Our previous studies revealed that the anti­ß2GPI Ab formed a stable oxidized low­density lipoprotein (oxLDL)/ß2GPI/anti­ß2GPI 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/ß2GPI/anti­ß2GPI Ab complex in human umbilical vein endothelial cells (HUVECs) in an attempt to determine the underlying mechanism. Reverse transcription­quantitative PCR, enzymy­linked immunosorbent assay, western blotting and immunofluorescence staining were performed to detect the expressions of inflammation related factors and adhesion molecules. Monocyte­binding assay was used to investigate the effects of oxLDL/ß2GPI/anti­ß2GPI Ab complex on monocyte adhesion to endothelial cells. The results demonstrated that the oxLDL/ß2GPI/anti­ß2GPI Ab complex upregulated the expression of Toll­like receptor (TLR)4 and the levels of NF­κB phosphorylation in HUVECs, and subsequently enhanced the expression levels of inflammatory cytokines, including TNF­α, IL­1ß and IL­6, 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/ß2GPI/anti­ß2GPI Ab complex in HUVECs were abolished by either TLR4 or NF­κB blockade. In conclusion, these findings suggested that the oxLDL/ß2GPI/anti­ß2GPI Ab complex may induce a hyper­inflammatory state in endothelial cells by promoting the secretion of proinflammatory cytokines and monocyte recruitment, which was discovered to be largely dependent on the TLR4/NK­κB signaling pathway.

Induction of tissue factor expression by anti-ß2-glycoprotein I is mediated by tumor necrosis factor α.

Antiphospholipid antibodies (aPL) are heterogeneous and there is evidence that binding specificity determines which cellular effects they can trigger. We have therefore hypothesised that the induction of tissue factor (TF) in monocytes and endothelial cells by aPL depends on their binding specificity. To further investigate this, we have analyzed the ability of three human monoclonal aPL with distinctly different binding specificities to induce transcription and cell surface expression of TF in monocytes and endothelial cells. Results with human monoclonal aPL were validated with IgG-fractions obtained from patients with APS. We confirmed previous results that a lipid reactive human monoclonal aPL rapidly induced TF transcription and cell surface expression in monocytes and endothelial cells. A monoclonal aPL reactive against ß2 glycoprotein I (ß2GPI) induced TF with a delayed time course. This was fully dependent on the induction of tumor necrosis factor alpha (TNFα) secretion as capture of TNFα by adalimumab prevented TF induction. This pattern was confirmed with patient IgG fractions. Both lipid reactive and anti-ß2GPI induced TF transcription. Unexpectedly, this activity of anti-ß2GPI was mediated fully by TNFα which was secreted in response to incubation with anti-ß2GPI. The role of TNFα in mediating TF induction by anti-ß2GPI may have wider implications for APS pathogenesis.

An in vitro model to mimic the thrombotic occlusion of small vessels in catastrophic antiphospholipid syndrome (CAPS).

Platelet activation and decrease in platelet count characterize the development of the most feared form of antiphospholipid syndrome (APS), i.e. catastrophic APS (CAPS). We aimed to assess if immuno-affinity purified anti-ß2-glycoprotein I (aß2GPI) antibodies enhance platelet activation inducing a significant flow obstruction in a platelet function analyzer (PFA). Affinity purified aß2GPI antibodies were obtained from 13 triple positive patients with a strong lupus anticoagulant (LA) and high titers of IgG anticardiolipin antibodies (aCL) and IgG aß2GPI. Platelet activation stimulated by adenosine diphosphate (ADP) in the presence or absence of aß2GPI was measured by the expression of P-selectin on platelet surface using flow cytometry. P-selectin expression remained close to baseline when normal whole blood was incubated with aß2GPI alone. When stimulated using aß2GPI combined with ADP, P-selectin expression (28.42 ± 5.15% vs. 20.98 ± 3.94%, p = 0.0076) was significantly higher than ADP alone. Closure time of normal whole blood passed through the PFA was significantly shorter using affinity purified aß2GPI than control IgG both in Col/ADP (160.1 ± 62.1 s vs. 218.6 ± 43.8 s; p = 0.021) and Col/EPI cartridges (149.5 ± 26.7 s vs. 186.9 ± 45.5 s; p = 0.030). Thus, platelet activation is enhanced by aß2GPI antibodies with a consequent premature closure in a PFA, possibly resembling that in microcirculation in patients with CAPS.

T cells from induced and spontaneous models of SLE recognize a common T cell epitope on ß2-glycoprotein I.

Systemic lupus erythematosus is a prototypic model for B-cell epitope spread in autoimmunity. Autoantibodies to numerous molecularly distinct self-antigens emerge in a sequential manner over several years, leading to disease manifestations. Among the earliest autoantibodies to appear are those targeting phospholipid-binding proteins, particularly ß2-glycoprotein I. Notably, mice immunized with ß2-glycoprotein I and lipopolysaccharide develop a strong T cell response to ß2-glycoprotein I that is associated with autoantibody production and renal disease, similar to that seen in human SLE. Here we hypothesized that mice with murine systemic lupus erythematosus, whether induced or spontaneous, should have T cells that recognize ß2-glycoprotein I. We evaluated the response of splenic T cells from mice with induced (C57BL/6 and C3H/HeN) and spontaneous (MRL/lpr) systemic lupus erythematosus to peptides spanning the entire sequence of human ß2GPI. We found that mice with induced and spontaneous systemic lupus erythematosus recognize a common T cell epitope (peptide 31; LYRDTAVFECLPQHAMFG) in domain III of ß2-glycoprotein I. ß2GPI-reactive CD4+ T cells from the two models differed primarily in cytokine production: T cells from mice with induced SLE expressed IFN-γ, while T cells from MRL/lpr mice expressed both IL-17 and IFN-γ, indicating that IL-17-expressing T cells are not necessary for generating a ß2GPI-reactive T cell response. These data suggest that the generation of a ß2-glycoprotein I-reactive T cell response is shared by both induced and spontaneous models of systemic lupus erythematosus and that this T cell response may mediate epitope spread to autoantibodies in both models.

Protective Effects of Reduced Beta 2 Glycoprotein I on Liver Injury in Streptozotocin (STZ)-Diabetic Rats by Activation of AMP-Activated Protein Kinase.

BACKGROUND Protective effects of reduced beta 2 glycoprotein I (Rb2GPI) against vascular injury of diabetes mellitus have been extensively investigated. However, the effects of Rb2GPI on liver injury in diabetic animals have not been reported. MATERIAL AND METHODS A diabetic rat model of was produced by systemic injection of streptozotocin (STZ). Rats were divided into a normal control group, a model group, and an Rb2GPI treatment group (N=6 in each group). After treatments, blood serum and liver tissue were collected to test the protection of Rb2GPI. AMP-activated protein kinase (AMPK) was detected by immunohistochemistry and Western blotting. RESULTS Our results revealed that Rß2GPI reduced blood glucose, serum creatinine, and urea nitrogen levels, as well as serum inflammation cytokines, including interleukin (IL)-6, tumor necrosis factor (TNF)-α and C-reactive protein in the diabetic rats. Importantly, Rß2GPI prevented liver injury in the diabetic rats as confirmed by hematoxylin-eosin (H&E) staining, alanine transaminase, aspartate transaminase, and gamma-glutamyl transferase. Reactive oxygen species (ROS) were promoted by diabetic modeling and were attenuated by Rß2GPI administration. Moreover, Rß2GPI significantly reduced liver catalase, malondialdehyde, and superoxide dismutase levels in the diabetic rats. Rß2GPI reduced liver glycolipid storage in STZ diabetic rats. Both immunohistochemistry and Western blotting demonstrated that Rß2GPI promoted AMPK phosphorylation in the diabetic rats. CONCLUSIONS Our data proved that Rß2GPI prevented liver injury in diabetic rats, likely through activating the AMPK signaling pathway.

Decreased hippocampal cell proliferation in mice with experimental antiphospholipid syndrome.

The antiphospholipid syndrome (APS) is an autoimmune disease characterized by the presence of antiphospholipid antibodies, which may trigger vascular thrombosis with consecutive infarcts. However, cognitive dysfunctions representing one of the most commonest neuropsychiatric symptoms are frequently present despite the absence of any ischemic brain lesions. Data on the structural and functional basis of the neuropsychiatric symptoms are sparse. To examine the effect of APS on hippocampal neurogenesis and on white matter, we induced experimental APS (eAPS) in adult female Balb/C mice by immunization with ß2-glycoprotein 1. To investigate cell proliferation in the dentate gyrus granular cell layer (DG GCL), eAPS and control mice (n = 5, each) were injected with 5-bromo-2'-deoxyuridine (BrdU) once a day for 10 subsequent days. Sixteen weeks after immunization, eAPS resulted in a significant reduction of BrdU-positive cells in the DG GCL compared to control animals. However, double staining with doublecortin and NeuN revealed a largely preserved neurogenesis. Ultrastructural analysis of corpus callosum (CC) axons in eAPS (n = 6) and control mice (n = 7) revealed no significant changes in CC axon diameter or g-ratio. In conclusion, decreased cellular proliferation in the hippocampus of eAPS mice indicates a limited regenerative potential and may represent one neuropathological substrate of cognitive changes in APS while evidence for alterations of white matter integrity is lacking.

Mutants of ß2-glycoprotein I: Their features and potent applications.

ß2-Glycoprotein I (ß2GPI) is a highly-glycosylated plasma protein composed of five homologous domains which regulates coagulation, fibrinolysis, and/or angiogenesis by interacting to negatively charged hydrophobic molecules and/or with plasminogen and its metabolites. The present study focused on structural and functional characterization of ß2GPI's domain I (DI) and V (DV). Through N-terminal amino acid sequencing, a novel plasmin-cleaved site at K287C288 was identified in DV. We further modified the intact DV by altering two amino acids at specific proteolytic cleavage sites to generate three stable DV mutants: DV(PP), (PE), and (AA). Results of both SDS-PAGE and MALDI-TOF-MS showed that all three DV mutants were more stable than the intact DV, and DV(PE) was predominantly resistant to proteolysis. Competitive ELISA assessed affinities of intact ß2GPI and those mutants to cardiolipin. In culture system, all DV and DI mutants potently inhibited HUVEC's proliferation by 18-30% as compared to control. Only DI and nicked ß2GPI showed significant inhibition in HUVEC's tube formation. Moreover, DV(PE)-coated affinity columns demonstrated its binding property towards anionic lipids and could substantially isolate anionic DOPS from zwitterionic DOPC as a purification model. In summary, the proteolytic resistant and unhindered phospholipid (PL) binding properties of DV(PE) have made it an appealing element for subsequent prospective studies. Future in-depth characterization and optimized applications of cleavage-resistant DV(PE) would complement its full capacity as a novel clinical modality in the field of vascular imaging and/or lipidomics studies.

The purification of reduced ß2-glycoprotein I showed its native activity in vitro.

BACKGROUND: New evidence has shown that reduced ß2-glycoprotein I (ß2GPI) has anti-oxidative stress and anti-inflammatory activity. However, the details are still poorly understood. This study aims to prepare stable reduced ß2GPI with its native bioactivity in vitro. METHODS: Human ß2GPI was purified from plasma first with perchloric acid precipitation and then purified with a series of chromatography methods including Sephadex G-25 desalting, SP HP, AF-heparin HC-650 M, and Sephacryl S-200. The purified human ß2GPI was reduced with thioredoxin-1 (TRX-1) activated by DL-dithiothreitol (DTT). Glutathione (GSH) was selected to block the free thiols in reduced ß2GPI. LC/MS was used to verify the location of free thiols. Western blot analysis was used to detect ß2GPI immunoreactivity. MTS and flow cytometry were conducted to investigate its biological effect on oxidative-stress-induced death of human umbilical vein endothelial cells (HUVECs). The levels of tumour necrosis factor-alpha (TNF-α),interleukin-6 (IL-6) interleukin-10 (IL-10),interleukin-12P70 (IL-12P70),interferon-gamma (IFN-γ) and monocyte chemoattractant protein -1(MCP-1) in mouse serum were quantified to assess its anti-inflammatory activity in lipopolysaccharide (LPS)-mediated systemic inflammation. RESULTS: We obtained approximately 10 mg ß2GPI (purity 98.7%) from 200 ml plasma. The protein yield was 0.05 mg/ml plasma. ß2GPI was then reduced by TRX-1/DTT in vitro; the free thiols were detected on Cys288 and Cys326 in domain V of ß2GPI. The GSH blockage stabilized the reduced ß2GPI in vitro. This reduced ß2GPI can be recognized by the anti-ß2GPI antibody, can significantly reduce the death of HUVECs after H2O2 treatment and can significantly decrease the levels of TNF-α, IL-6,IFN-γ and MCP-1 in mice upon LPS stimulation. CONCLUSION: Stable reduced ß2GPI can be obtained in vitro by TRX-1 deoxidation followed by the blockage of thiols with GSH. This reduced ß2GPI maintains the same immunological activity as oxidized ß2GPI and has the ability to counter the oxidative stress induced by H2O2 in HUVECs and inflammation in LPS-mediated inflammation in mice.

Effects of reduced ß2 glycoprotein I on high glucose­induced cell death in HUVECs.

Reduced ß2 glycoprotein I (ß2GPI) has been demonstrated to exhibit a beneficial effect in diabetic atherosclerosis and retinal neovascularization. However, the effect of reduced ß2GPI on vascular disorders in diabetic mellitus (DM) remains to be elucidated. The present study established a high glucose­induced injury model using human umbilical cords veins (HUVECs) and evaluated the protective effects of reduced ß2GPI against the injury. The data demonstrated that a low concentration of reduced ß2GPI (0.5 µM) mitigated high glucose­induced cell loss, decreased nitric oxide (NO) production and resulted in calcium overloading. Mechanically, reduced ß2GPI additionally reversed high glucose­induced phosphatase and tensin homolog (PTEN) accumulation, decrease of protein kinase B phosphorylation and nitric oxide synthase activity, and increase of cyclooxygenase­2 activity. It was further confirmed that PTEN inhibitor­bpV (1 µM) exhibited similar effects to those resulting from reduced ß2GPI. Overall, the data revealed that reduced ß2GPI exerts protective effects from glucose­induced injury in HUVECs, potentially via decreasing PTEN levels. The present study suggests reduced ß2GPI may act as a novel therapeutic strategy for the treatment of vascular disorders in DM.

[The oxLDL/ß2GPI/ß2GPI-Ab complex promotes the migration and the expression of inflammatory cytokines in human umbilical vein endothelial cells].

Objective To study the effects of the complex of oxidized low density lipoprotein/ß2-glycoprotein I/ß2-glycoprotein I antibodies (oxLDL/ß2GPI/ß2GPI-Ab) on the migration of human umbilical vein endothelial cells (HUVECs) and the expression of inflammatory cytokines, and their underlying Toll-like receptor (TLR4) pathway. Methods HUVECs were treated with oxLDL, oxLDL/ß2GPI complex, oxLDL/ß2GPI-Ab complex, oxLDL/ß2GPI/ß2GPI-Ab complex, or lipopolysaccharide (LPS) for a period of time in their corresponding groups. The migration of HUVECs was observed by the wound-healing assay. The mRNA and protein levels of TLR4 in HUVECs were detected by real-time quantitative PCR (qRT-PCR) and Western blotting, respectively. The cells were pretreated with or without TAK-242 (the inhibitor of TLR4) 2 hours before stimulated by corresponding stimulus as described above. Then, the contents of monocyte chemotactic protein-1 (MCP-1), interleukin 1ß (IL-1ß) and IL-6 in cell culture supernatant were determined by ELISA, and their mRNAs were detected by qRT-PCR. Results The oxLDL/ß2GPI/ß2GPI-Ab complex promoted the migration of HUVECs effectively, and increased the expression of TLR4. The oxLDL/ß2GPI/ß2GPI-Ab complex increased the expressions of MCP-1, IL-1ß, and IL-6. TAK-242 could reduce the effects of oxLDL/ß2GPI/ß2GPI-Ab complex. Conclusion The oxLDL/ß2GPI/anti-ß2GPI-Ab complex can promote the migration of HUVECs and the expression of related inflammatory cytokines, and TLR4 may be involved in this process.

[The oxLDL/ß2GPI/anti-ß2GPI antibody complex promotes A7r5 chemotaxis, migration and lipid accumulation].

Objective To explore the effect of the oxidized low-density lipoprotein/ß2 -glycoprotein I/ß2 glycoprotein I antibody (oxLDL/ß2GPI/ß2GPI-Ab) complex on the migration, chemotaxis and lipid accumulation of rat thoracic aortic smooth muscle A7r5 cell line, and unveil the role of Toll-like receptor 4 (TLR4) pathway during the process. Methods A7r5 cells were cultured in vitro with or without the pretreatment of TAK-242, a TLR4 inhibitor. Then the cells were stimulated by oxLDL, oxLDL/ß2GPI complex, ß2GPI, ß2GPI-Ab, ß2GPI/ß2GPI-Ab complex or oxLDL/ß2GPI/ß2GPI-Ab complex. Total RNA was extracted from the cells and the mRNA levels of monocyte chemoattractant protein-1 (MCP-1), matrix metalloproteinase-9 (MMP-9) and acetyl-CoA acetyltransferase 1 (ACAT1) were evaluated by real-time quantitative PCR. The cell supernatants were collected, and the protein levels of MCP-1 and MMP-9 were determined by ELISA. The migration of A7r5 was observed through wound-healing test. A7r5 cells pretreated with or without TAK-242 were stimulated by oxLDL or oxLDL/ß2GPI/ß2GPI-Ab complex, and the content of total cholesterol (TC) and free cholesterol (FC) in them were measured by corresponding test kits. Then the level of intracellular cholesterol ester (CE) was calculated. Results The oxLDL/ß2GPI/ß2GPI-Ab complex promoted the migration and cholesterol accumulation, and up-regulated the expressions of MCP-1, MMP-9 and ACAT1 in A7r5 cells. In addition, the application of TLR4 inhibitor, TAK-242, suppressed these effects. Conclusion oxLDL/ß2GPI/ß2GPI-Ab complex contributes to the process of atherosclerosis through promoting the migration, chemotaxis and lipid accumulation of a7r5 in a TLR4-dependent manner.

ß2-Glycoprotein I Inhibits Vascular Endothelial Growth Factor-Induced Angiogenesis by Suppressing the Phosphorylation of Extracellular Signal-Regulated Kinase 1/2, Akt, and Endothelial Nitric Oxide Synthase.

Angiogenesis is the process of new blood vessel formation, and it plays a key role in various physiological and pathological conditions. The ß2-glycoprotein I (ß2-GPI) is a plasma glycoprotein with multiple biological functions, some of which remain to be elucidated. This study aimed to identify the contribution of 2-GPI on the angiogenesis induced by vascular endothelial growth factor (VEGF), a pro-angiogenic factor that may regulate endothelial remodeling, and its underlying mechanism. Our results revealed that ß2-GPI dose-dependently decreased the VEGF-induced increase in endothelial cell proliferation, using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and the bromodeoxyuridine (BrdU) incorporation assays. Furthermore, incubation with both ß2-GPI and deglycosylated ß2-GPI inhibited the VEGF-induced tube formation. Our results suggest that the carbohydrate residues of ß2-GPI do not participate in the function of anti-angiogenesis. Using in vivo Matrigel plug and angioreactor assays, we show that ß2-GPI remarkably inhibited the VEGF-induced angiogenesis at a physiological concentration. Moreover, ß2-GPI inhibited the VEGF-induced phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), Akt, and endothelial nitric oxide synthase (eNOS). In summary, our in vitro and in vivo data reveal for the first time that ß2-GPI inhibits the VEGF-induced angiogenesis and highlights the potential for ß2-GPI in anti-angiogenic therapy.

The Fifth Domain of Beta 2 Glycoprotein I Protects from Natural IgM Mediated Cardiac Ischaemia Reperfusion Injury.

Reperfusion after a period of ischemia results in reperfusion injury (IRI) which involves activation of the inflammatory cascade. In cardiac IRI, IgM natural antibodies (NAb) play a prominent role through binding to altered neoepitopes expressed on damaged cells. Beta 2 Glycoprotein I (ß2GPI) is a plasma protein that binds to neoepitopes on damaged cells including anionic phospholipids through its highly conserved Domain V. Domain I of ß2GPI binds circulating IgM NAbs and may provide a link between the innate immune system, IgM NAb binding and cardiac IRI. This study was undertaken to investigate the role of Β2GPI and its Domain V in cardiac IRI using wild-type (WT), Rag-1 -/- and ß2GPI deficient mice. Compared with control, treatment with Domain V prior to cardiac IRI prevented binding of endogenous ß2GPI to post-ischemic myocardium and resulted in smaller myocardial infarction size in both WT and ß2GPI deficient mice. Domain V treatment in WT mice also resulted in less neutrophil infiltration, less apoptosis and improved ejection fraction at 24 h. Rag-1 -/- antibody deficient mice reconstituted with IgM NAbs confirmed that Domain V prevented IgM NAb induced cardiac IRI. Domain V remained equally effective when delivered at the time of reperfusion which has therapeutic clinical relevance.Based upon this study Domain V may function as a universal inhibitor of IgM NAb binding in the setting of cardiac IRI, which offers promise as a new therapeutic strategy in the treatment of cardiac IRI.

Do Beta 2-Glycoprotein I Disulfide Bonds Protect the Human Retina in the Setting of Age-Related Macular Degeneration?

Age-related macular degeneration (AMD) affects the region of the retina that is responsible for high-resolution vision. It is a major cause of blindness in the aging population. This is the first study that examines the association of redox-modified, cysteine-based, post-translational forms of beta 2-glycoprotein I (ß2GPI) in the plasma of individuals with early and late stages of patients with AMD compared with controls. Exploration is also undertaken to assess whether the free thiol form of ß2GPI versus the oxidized disulfide form have distinct functional properties in the setting of hydrogen peroxide (H(2)O(2))-mediated cell death of an immortalized human retinal pigment epithelium (RPE) cell line. We demonstrate ß2GPI in the retina and choroid of patients with AMD. Free thiol ß2GPI is shown to protect the immortalized human RPE cell line against H(2)O(2)-induced cell death, whereas the oxidized form of ß2GPI and free thiol bovine serum albumin were not protective. Free thiol ß2GPI levels were significantly decreased in patients with late AMD compared with early AMD and healthy controls. Our observations lead to the hypothesis that free thiol ß2GPI may protect against oxidative stress injury to RPE cells in the early stages of AMD.

Lipopolysaccharide Enhances Beta2-Glycoprotein I Activation of Nuclear Factor κB in Liver Cancer Cells.

BACKGROUND: Beta2-glycoprotein I (ß2GPI) is a highly abundant glycoprotein in plasma. Our previous study demonstrated strong ß2GPI expression in hepatitis B-related hepatocellular carcinoma (HCC) tissue and the combination of ß2GPI and hepatitis B surface antigen (HBsAg) was shown to significantly activate the nuclear factor kappa B (NF-κB). To investigate whether lipopolysaccharide (LPS) enhances ß2GPI activation of NF-ßB and the expression of downstream factors (e.g., tumor necrosis factor alpha, TNF-α; interleukin-1 beta, IL-1ß; alpha-fetoprotein, AFP) in the human hepatoma cell line, SMMC-7721. METHODS: Experimental samples were divided into 4 groups as follows: Group A--blank cell group (SMMC-7721); group B--low, medium, and high LPS concentration groups (1 ng/mL; 10 ng/mL; and 100 ng/mL, respectively); group C--ß2GPI transfected group; and group D--ß2GPI + low, medium, or high concentrations from the LPS affected group. Activation of NF-κB was evaluated using laser scanning confocal microscopy. Expression of downstream factors was measured by ELISA. RESULTS: Degrees of NF-κB activation in groups B, C, and D were varied. NF-κB activation in group D was the most significant, and the expressions of downstream factors, TNF-α and IL-1ß, were the highest level of activation among the groups (p < 0.05), showing an LPS dose-dependency. CONCLUSIONS: LPS enhanced the signal transduction of ß2GPI in liver cancer cells leading to activation of NF-κB, which triggered downstream signal transduction and increased the expression of downstream factors. This suggests that LPS enhancement of ß2GPI signal transduction may play a role in promoting the development of liver cancer.

Reduced beta 2 glycoprotein I improves diabetic nephropathy via inhibiting TGF-ß1-p38 MAPK pathway.

PURPOSE: Beta 2 glycoprotein I (ß2GPI) has been shown the positive effect on diabetic atherosclerosis and retinal neovascularization. ß2GPI can be reduced by thioredoxin-1, resulting in the reduced state of ß2GPI. The possible protective effects of ß2GPI and reduced ß2GPI on diabetic nephropathy (DN) are not fully elucidated. The purpose of this study was to test a hypothesis that ß2GPI and reduced ß2GPI would improve DN in streptozotocin (STZ) induced diabetic mice and high-glucose (HG) exposed rat mesangial cell (RMC). METHODS: The STZ-induced Balb/c mice and HG exposed RMCs were administrated with ß2-GPI and reduced ß2-GPI at different time and concentrations gradient respectively. The changes of glomerular structure and expression of collagen IV, TGF-ß1, p38 MAPK and phospho-p38 MAPK in renal cortical and mesangial cells were observed by immunohistochemical techniques, quantitative real-time PCR and western blot with or without the treatment of ß2-GPI and reduced ß2-GPI. RESULTS: ß2GPI and reduced ß2GPI improved early clinical and pathological changes of DN in STZ-diabetic mice. Treatment with ß2GPI and reduced ß2GPI in the STZ-diabetic mice and HG exposed RMCs resulted in decrease expression levels of TGF-ß1 and collagen IV, with concomitant decrease in phospho-p38 MAPK expression. CONCLUSIONS: ß2GPI and reduced ß2GPI improved renal structural damage and kidney function. The renoprotective and antifibrosis effects of ß2GPI and reduced ß2GPI on DN were closely associated with suppressing the activation of the TGF-ß1-p38 MAPK pathway.

Domain I-IV of ß2-glycoprotein I inhibits advanced glycation end product-induced angiogenesis by down-regulating vascular endothelial growth factor 2 signaling.

Advanced glycation end products (AGEs) are a contributing factor in the angiogenesis that is characteristic of proliferative diabetic retinopathy. However, a previous study made a promising observation that domain I­IV of ß2­glycoprotein I (DI­IV) inhibits angiogenesis in human umbilical vein cells. The present study aimed to confirm the inhibition of AGE­induced angiogenesis in retinal endothelial cells by DI­IV and to investigate the potential underlying mechanisms. The RF/6A rhesus macaque choroid­retinal vascular endothelial cell line was cultured in vitro and treated with AGEs in the presence or absence of different concentrations of DI­IV. The proliferation, migration and tube formation of the RF/6A cells were evaluated using MTS assays, in vitro wound healing assays and in vitro Matrigel angiogenesis assays, respectively. The mRNA expression of vascular endothelial growth factor (VEGF), VEGF receptor (VEGFR) 2, VEGFR 1 and receptor for AGE (RAGE) were quantified by reverse transcription quantitative polymerase chain reaction. The expression of VEGFR­1, VEGFR­2 and the activation of protein kinase B (Akt) and extracellular signal­regulated kinase (ERK) were also assessed by western blot analysis. The results indicated that AGEs promoted the migration, proliferation and tube formation of RF/6A cells in vitro (P<0.05), increased the expression of VEGF, VEGFR­2 and RAGE (P<0.05) and increased the phosphorylation of Akt and ERK (P<0.05). DI­IV inhibited the increase in VEGFR­2 mRNA and protein, but did not inhibit the increase in VEGF or RAGE mRNAs. These results led to the conclusion that DI­IV inhibited AGE­induced angiogenesis in the RF/6A cells, which was accompanied by a downregulation in the expression of VEGFR­2 and its downstream phosphatidylinosol 3­kinase/Akt and mitogen­activated protein kinase/ERK1/2 pathways. These findings provide further support towards the treatment of proliferative diabetic retinopathy by interventions that act via a mechanism similar to that of DI­IV.