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.

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.

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.

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.

Subclinical atherosclerosis in systemic lupus erythematosus and antiphospholipid syndrome: focus on ß2GPI-specific T cell response.

OBJECTIVE: Systemic Lupus Erythematosus (SLE) and antiphospholipid syndrome (APS) are associated with a high prevalence of atherosclerosis. ß2 glycoprotein I (ß2GPI) represents a link between autoimmunity and endothelial dysfunction. Recently, ß2GPI reactive T cells have been identified; however, their role in atherosclerosis is still under investigation. We evaluated early atherosclerosis in patients with SLE and APS and investigated T cell reactivity to ß2GPI and its relationship with atherosclerotic process. APPROACH AND RESULTS: Fifty SLE, 18 patients with primary APS (PAPS), and 25 healthy controls were enrolled. Demographic and clinical data, including traditional cardiovascular risk factors, were recorded. Monocyte ß2GPI and Tissue Factor (TF) expression and peripheral blood mononuclear cell response to ß2GPI stimulation were evaluated. Doppler ultrasound was performed to investigate flow-mediated dilatation (FMD) and carotid intima-media thickness (IMT). We detected an increase in mean IMT and a decrease in FMD in patients with SLE versus controls (P<0.05 and P=0.0001, respectively) and a decrease in FMD in patients with PAPS versus controls (P<0.05). Monocyte ß2GPI and TF expression was higher in patients with SLE and PAPS than in controls (P=0.006 and P=0.001, respectively); no correlation of monocyte ß2GPI and TF with IMT or FMD was detected. ß2GPI induced peripheral blood mononuclear cell proliferation in 32% of patients with SLE, 25% of patients with PAPS yet in none of the controls. Proliferative response to ß2GPI correlated with a history of arterial thrombosis, thrombocytopenia, and IMT >0.9 mm. CONCLUSIONS: A significant percentage of patients with SLE and PAPS show a ß2GPI-specific T cell reactivity, which is associated with subclinical atherosclerosis.

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.

Tolerogenic dendritic cells specific for ß2-glycoprotein-I Domain-I, attenuate experimental antiphospholipid syndrome.

Tolerogenic dendritic cells (tDCs) have the potential to control the outcome of autoimmunity by modulating the immune response. The aim of this study was to uncover the tolerance efficacy attributed to beta-2-glycoprotein-I (ß2GPI) tDCs or ß2GPI domain-I (D-I) and domain-V (D-V)-tDCs in mice with antiphospholipid syndrome (APS). tDCs were pulsed with ß2GPI or D-I or D-V derivatives. Our results revealed that ß2GPI related tDCs phenotype includes CD80(high), CD86(high) CD40(high) MHC class II(high). The miRNA profiling encompass miRNA 23b(high), miRNA 142-3p(low) and miRNA 221(low). In addition the ß2GPI related tDCs showed reduced secretion of IL-1ß, IL-12 and IL-23. D-I tDCs treatment was more efficient than ß2GPI tDCs in inducing of tolerance in APS mice, manifested by lowered titers of anti- ß2GPI antibodies (Abs) and reduced percentage of fetal loss. Tolerance induction was accompanied by poor T cell response to ß2GPI, high numbers of CD4 + CD25 + FOXP3 + T-regulatory cells (Treg), reduced levels of IFNγ, IL-17 and increased expression of IL-10 and TGFß. Tolerance was successfully transferred by Treg cells from the tolerized mice to ß2GPI immunized mice. We conclude that predominantly D-I-tDCs and ß2GPI tDCs have the potential to attenuate experimental APS by induction of Treg cells, reduction of anti- ß2GPI Abs titers and increased expression of anti-inflammatory cytokines. We suggest that ß2-GPI-D-I-tDCs may offer a novel approach for developing therapy for APS patients.

Small ß2-glycoprotein I peptides protect from intestinal ischemia reperfusion injury.

Intestinal ischemic events, which are followed by reperfusion, induce significant tissue damage and frequently result in multiple organ failure, with >70% mortality. Upon reperfusion, excessive inflammation leads to exacerbated tissue damage. Previous studies indicated that binding of the serum protein, ß2-glycoprotein I, to the endothelium initiates a cascade of inflammatory molecules that is required for damage. We hypothesized that peptides derived from the binding domain (domain V) of ß2-glycoprotein I would attenuate ischemia/reperfusion-induced damage and inflammation in a therapeutic manner. Using a mouse model of intestinal ischemia/reperfusion, we administered peptides either prior to ischemia or at clinically relevant time points during reperfusion and evaluated intestinal tissue damage and inflammation after 2 h of reperfusion. We demonstrate that multiple peptides attenuate injury and inflammation in a dose-dependent manner and, perhaps more significantly, are efficacious when administered up to 30 min after the onset of reperfusion. In addition, an all D-amino acid retro-inverso peptide was biologically active. Thus, the ß2-glycoprotein I-derived peptides attenuate injury and inflammation when administered in a therapeutic manner in intestinal ischemia/reperfusion injury.

Effects of reduced ß2-glycoprotein I on the expression of aortic matrix metalloproteinases and tissue inhibitor matrix metalloproteinases in diabetic mice.

BACKGROUND: Reduced ß(2)-glycoprotein I (reduced ß(2)GP I), which has free sulfhydryl groups, is present in plasma and serum; it can protect vascular endothelial cells from damage due to oxidative stress in vitro. We investigated the effects of reduced ß(2)GP I on the expression of various matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) in the aortas of diabetic mice. METHODS: We provided 120 female 8-week-old Balb/c mice with a high sugar, high fat diet. After 8 weeks they were injected with streptozotocin to induce diabetes. We treated mice in the mono dose groups with ß(2)GP I, reduced ß(2)GP I, or phosphate-buffered saline (PBS) on day 1 and fed them for 3 weeks. The mice in the complex dose groups were treated with ß(2)GP I, reduced ß(2)GP I, or PBS on days 1 and 22 and fed for 6 weeks. Control mice were given a standard chow diet. Blood lipids were measured at the end of 3 or 6 weeks, and aortas removed to observe morphological and molecular biological changes. RESULTS: The low-density lipoprotein cholesterol levels in mice of the reduced ß(2)GP I group were lower than those in the diabetic group. Aortic lipid deposition in the reduced ß(2)GP I group was significantly less than in the diabetic control group. In the aortas, reduced ß(2)GP I decreased MMP2/TIMP2 mRNA and protein expression levels, and MMP9/TIMP1 expression levels compared with those in diabetic controls. Reduced ß(2)GP I down-regulated p38 mitogen-activated protein kinase (p38MAPK) mRNA expression and phosphorylated p38MAPK protein expression compared with those in diabetic controls of the complex dose group. CONCLUSIONS: Reduced ß(2)GP I plays a role in diabetic mice related to vascular protection, inhibiting vascular lipid deposition, and plaque formation by reducing MMPs/TIMPs expression through down-regulation of the p38MAPK signaling pathway.

C-reactive protein/oxidised low-density lipoprotein/ß2-glycoprotein I complex promotes atherosclerosis in diabetic BALB/c mice via p38mitogen-activated protein kinase signal pathway.

BACKGROUND: The aim of this study was to investigate the effect of C-reactive protein/oxidised low-density lipoprotein/ß2-glycoprotein I (CRP/oxLDL/ß2GPI) complex on atherosclerosis (AS) in diabetic BALB/c mice. METHODS: BALB/c mice were fed high-fat and normal diet. Eight weeks later, the mice fed with high-fat diet were injected with streptozotocin (STZ) to induce diabetes. The diabetic mice were respectively injected twice monthly with 20 µg oxLDL, 20 µg ß2GPI, 40 µg oxLDL/ß2GPI complex, 44 µg CRP/oxLDL/ß2GPI complex, and PBS. Aortas were stained with Sudan IV to investigate lipid plaque formation. The infiltration condition of smooth muscle cells (SMCs), macrophages, and T cells in the aortas were determined by immunohistochemistry (IH). The mRNA expressions of receptors associated with lipid metabolism were quantified by real-time PCR. The phosphorylation of p38 mitogen-activated protein kinase (p38MAPK) and MKK3/6 in aorta tissues were assessed by Western blot. The expression of inflammation cytokines was evaluated by protein chip. RESULTS: The lipid plaques were more extensive, the lumen area was obviously narrower, the ratio of intima and media thickness were increased, and the normal internal elastic lamia structure and endothelial cell disappeared (P < 0.05) in the oxLDL and CRP/oxLDL/ß2GPI groups (P < 0.05). CRP/oxLDL/ß2GPI complex dramatically promoted infiltration of SMCs, macrophages, and T cells, improved the mRNA expression of ABCA1 and ABCG1, but reduced the mRNA expression of SR-BI and CD36 and increased the phosphorylation of p38MAPK and MKK3/6 (all P < 0.05). The highest expression levels of IL-1, IL-9, PF-4, bFGF, and IGF-II were detected in the CRP/oxLDL/ß2GPI group (P < 0.05). CONCLUSIONS: CRP/oxLDL/ß2GPI complex aggravated AS in diabetic BALB/c mice by increasing lipid uptake, the mechanism of which may be mediated by the p38MAPK signal pathway.

Reduced beta2-glycoprotein I protects macrophages from ox-LDL-induced foam cell formation and cell apoptosis.

BACKGROUND: Reduced beta2-glycoprotein I (beta2-GPI) is a free thiol-containing form of beta2-GPI that displays a powerful effect in protecting endothelial cells from oxidative stress-induced cell death. The present study aims to investigate the effect of beta2-GPI or reduced beta2-GPI on ox-LDL-induced foam cell formation and on cell apoptosis and to determine the possible mechanisms. METHODS: The RAW264.7 macrophage cell line was selected as the experimental material. Oil red O staining and cholesterol measurement were used to detect cholesterol accumulation qualitatively and quantitatively, respectively. Flow cytometry was used to detect cell apoptosis. Real-time quantitative PCR was used to detect the mRNA expression of the main proteins that are associated with the transport of cholesterol, such as CD36, SRB1, ABCA1 and ABCG1. Western blot analysis was used to detect the protein expression of certain apoptosis-related proteins, such as caspase-9, caspase-3, p38 MAPK/p-p38 MAPK and JNK/p-JNK. RESULTS: Beta2-GPI or reduced beta2-GPI decreased ox-LDL-induced cholesterol accumulation (96.45 ± 8.51 µg/mg protein vs. 114.35 ± 10.38 µg/mg protein, p < 0.05;74.44 ± 5.27 µg/mg protein vs. 114.35 ± 10.38 µg/mg protein, p < 0.01) and cell apoptosis (30.00 ± 5.10% vs. 38.70 ± 7.76%, p < 0.05; 20.66 ± 2.50% vs. 38.70 ± 7.76%, p < 0.01), and there are significant differences between beta2-GPI and reduced beta2-GPI (p < 0.05). Reduced beta2-GPI decreased the ox-LDL-induced expression of CD36 mRNA and ABCA1 mRNA (p < 0.05), as well as CD36, cleaved caspase-9, cleaved caspase-3, p-p38 MAPK and p-JNK proteins (p < 0.05 or p < 0.01). Beta2-GPI did not significantly decrease the expression of ABCA1 mRNA and the p-p38 MAPK protein. CONCLUSIONS: Both beta2-GPI and reduced beta2-GPI inhibit ox-LDL-induced foam cell formation and cell apoptosis, and the latter exhibits a stronger inhibition effect. Both of these glycoproteins reduce the lipid intake of macrophages by downregulating CD36 as well as protein expression. Reduced beta2-GPI inhibits cell apoptosis by reducing the ox-LDL-induced phosphorylation of p38 MAPK and JNK, and the amount of cleaved caspase-3 and caspase-9. Beta2-GPI does not inhibit the ox-LDL-induced phosphorylation of p38 MAPK.

{beta}2 Glycoprotein I ({beta}2GPI) binds platelet factor 4 (PF4): implications for the pathogenesis of antiphospholipid syndrome.

Antiphospholipid syndrome (APS) is an autoimmune thrombophilia characterized by arterial/venous thrombosis and/or pregnancy morbidity in the presence of antiphospholipid antibodies that mainly recognize beta2 glycoprotein I (beta2GPI). To investigate potential platelet ligands of beta2GPI, platelet membrane proteins from healthy persons and patients with APS were passed through a beta2GPI-affinity column. By using mass spectrometry, platelet factor 4 (PF4) appeared as the dominant beta2GPI binding protein. PF4 could bind in vitro, with high-affinity, recombinant beta2GPI, and the binding was abrogated by soluble beta2GPI. Coprecipitation experiments further confirmed this interaction. In silico molecular docking showed that PF4 tetramers can bind 2 beta2GPI molecules simultaneously. Size exclusion chromatography confirmed that anti-beta2GPI antibodies selectively interact with complexes composed of (beta2GPI)(2)-(PF4)(4). In addition, as shown by the beta2GPI antigenicity evaluation, the reactivity of APS sera was higher against PF4-beta2GPI complex than against beta2GPI alone. On complex formation, anti-beta2GPI-beta2GPI-PF4 significantly induced platelet p38MAPK phosphorylation and TXB2 production, mainly through F(ab')(2) fragments of antibodies. In summary, this study makes evident that beta2GPI forms stable complexes with PF4, leading to the stabilization of beta2GPI dimeric structure that facilitates the antibody recognition. This interaction can probably be involved in the procoagulant tendency of APS.

In vitro effect of anti-ß2 glycoprotein I antibodies on P-selectin expression, a marker of platelet activation.

OBJECTIVE: Antiphospholipid antibodies (aPL) associated with thrombembolic events and/or pregnancy morbidity characterize the so-called antiphospholipid syndrome (APS). Beta2glycoprotein I (ß2GPI) represents the major target antigen for aPL, but the pathogenic role of anti-ß2GPI antibodies (aß2GPI) is still unclear. Some authors assume they play a role in activating platelets. The effects of aß2GPI antibodies on platelet P-selectin expression were evaluated in this study. METHODS: Aß2GPI antibodies in the plasma of a pregnant APS patient were isolated by affinity chromatography during two different stages (catastrophic and quiescent) of the disease. Gel filtered platelets (100,000/µl) from healthy volunteers were incubated with ß2-GPI (20 µg/ml) and with different concentrations (5, 25 e 50 µg/ml) of aß2GPI antibodies. P-selectin surface expression on platelets was assessed by flow cytometry using a specific fluorescent antibody directed against P-selectin. RESULTS: Aß2GPI antibodies induced platelet activation only in the presence of thrombin receptor activator for peptide 6 (TRAP-6), a platelet agonist, at a subthreshold concentration. Aß2GPI antibody enhancement on platelet surface P-selectin expression was stronger in the catastrophic than in the quiescent phase of the disease (47% versus 15%). CONCLUSIONS: TRAP-6 dependent platelet activation by aß2GPI antibodies is consistent with the "two hit" pathogenetic hypothesis for thrombosis. Aß2GPI antibodies induce higher platelet P-selectin expression during the active rather than in the acute phases.

ß2-Glycoprotein-I based peptide regulate endothelial-cells tissue-factor expression via negative regulation of pGSK3ß expression and reduces experimental-antiphospholipid-syndrome.

Antiphospholipid syndrome (APS) is characterized by thromboembolic phenomena and recurrent fetal loss associated with elevated circulating anti-phospholipid/beta2glycoprotein-I(ß2GPI)-binding-antibodies(Abs). Individual APS patients harbor diverse clusters of circulating anti-ß2GPI Abs, targeting different epitopes on the ß2GPI molecule. Our novel approach was to construct a peptide composed of ß2GPI-ECs-binding-site (phospholipids-membrane), named "EMBI". EMBI exert dual activities: a) At first EMBI prevented ß2GPI ECs binding, thus reduced by 89% the binding of ß2GPI/anti-ß2GPI to the cells in comparison with 9.3% inhibition by EMBI scrambled form (scEMBI). b) Longer exposure of ECs to EMBI resulted in intracellular EMBI penetration which did not prevent ß2GPI/anti-ß2GPI binding to HUVEC. Surprisingly, ß2GPI/anti-ß2GPI did not activate ECs harboring EMBI, illustrated by prevention of E-selectin and tissue factor (TF) expression. The inhibition of TF mRNA transcription was illustrated by quantitative RT-PCR. EMBI decreased the expression of phosphorylated JNK1/2, p38, HSP27 and enhanced phosphorylation of glycogen synthase kinase-3ß (pGSK3ß). Knocking down the GSK3ß expression by siRNA-GSK3ß, reduced the TF expression by ß2GPI/anti-ß2GPI-exposed-HUVEC. In-vivo, EMBI significantly decreased the percentage of fetal loss in naïve mice infused with anti-ß2GPI Abs, p<0.04. Thus, the dual activity of EMBI may introduce EMBI as a potential novel candidate peptide, to treat patients with APS.

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.

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.

Agglutination of like-charged red blood cells induced by binding of beta2-glycoprotein I to outer cell surface.

Plasma protein-mediated attractive interaction between membranes of red blood cells (RBCs) and phospholipid vesicles was studied. It is shown that beta(2)-glycoprotein I (beta(2)-GPI) may induce RBC discocyte-echinocyte-spherocyte shape transformation and subsequent agglutination of RBCs. Based on the observed beta(2)-GPI-induced RBC cell shape transformation it is proposed that the hydrophobic portion of beta(2)-GPI molecule protrudes into the outer lipid layer of the RBC membrane and increases the area of this layer. It is also suggested that the observed agglutination of RBCs is at least partially driven by an attractive force which is of electrostatic origin and depends on the specific molecular shape and internal charge distribution of membrane-bound beta(2)-GPI molecules. The suggested beta(2)-GPI-induced attractive electrostatic interaction between like-charged RBC membrane surfaces is qualitatively explained by using a simple mathematical model within the functional density theory of the electric double layer, where the electrostatic attraction between the positively charged part of the first domains of bound beta(2)-GPI molecules and negatively charged glycocalyx of the adjacent RBC membrane is taken into account.