Endoplasmic reticulum stress occurs in association with the extrusion of toxic extracellular vesicles from human placentae treated with antiphospholipid antibodies.

Antiphospholipid autoantibodies (aPLs), a major maternal risk factor for preeclampsia, are taken into the syncytiotrophoblast where they bind intracellular vesicles and mitochondria. Subsequently, large quantities of extracellular vesicles (EVs) extruded from syncytiotrophoblast into the maternal circulation are altered such that they cause maternal endothelial cell activation. However, the mechanism driving this change is unknown. First trimester placental explants were treated with aPL for 18 h. The EVs were then collected by different centrifugation. The levels of HSP 70, misfolded proteins, caspase 8 activity, and Mixed Lineage Kinase domain-Like (MLKL) were measured in placental explants and EVs. In addition, the levels of TNF-α and CD95 in conditioned medium were also measured. Treating placental explants with aPL caused an increase in levels of HSP 70, misfolded proteins and MLKL in placental explants and EVs. Increased activity of caspase 8 was also seen in placental explants. Higher levels of TNF-α were seen conditioned medium from aPL-treated placental explant cultures. aPLs appear to induce endoplasmic reticulum stress in the syncytiotrophoblast in a manner that involved caspase 8 and TNF-α. To avoid accumulation of the associated misfolded proteins and MLKL, the syncytiotrophoblast exports these potentially dangerous proteins in EVs. It is likely that the dangerous proteins that are loaded into placental EVs in preeclampsia contribute to dysfunction of the maternal cells.

Hydroxychloroquine inhibits proinflammatory signalling pathways by targeting endosomal NADPH oxidase.

OBJECTIVES: Hydroxychloroquine (HCQ) has been used for decades to treat patients with rheumatic diseases, for example, systemic lupus erythematosus (SLE), rheumatoid arthritis or the antiphospholipid syndrome (APS). We hypothesise that HCQ might target endosomal NADPH oxidase (NOX), which is involved in the signal transduction of cytokines as well as antiphospholipid antibodies (aPL). METHODS: For in vitro experiments, monocytic cells were stimulated with tumour necrosis factor α (TNFα), interleukin-1ß (IL-1ß) or a human monoclonal aPL and the activity of NOX was determined by flow cytometry. The expression of genes known to be induced by these stimuli was quantified by quantitative reverse transcription PCR. Live cell imaging was performed by confocal laser scanning microscopy. Finally, the effects of HCQ on NOX-induced signal transduction were analysed in an in vivo model of venous thrombosis. RESULTS: HCQ strongly reduces or completely prevents the induction of endosomal NOX by TNFα, IL-1ß and aPL in human monocytes and MonoMac1 cells. As a consequence, induction of downstream genes by these stimuli is reduced or abrogated. This effect of HCQ is not mediated by direct interference with the agonists but by inhibiting the translocation of the catalytic subunit of NOX2 (gp91phox) into the endosome. In vivo, HCQ protects mice from aPL-induced and NOX2-mediated thrombus formation. CONCLUSIONS: We describe here a novel mechanism of action of HCQ, that is, interference with the assembly of endosomal NOX2. Since endosomal NOX2 is involved in many inflammatory and prothrombotic signalling pathways, this activity of HCQ might explain many of its beneficial effects in rheumatic diseases including the APS.

Proof-of-concept study demonstrating the pathogenicity of affinity-purified IgG antibodies directed to domain I of ß2-glycoprotein I in a mouse model of anti-phospholipid antibody-induced thrombosis.

OBJECTIVE: IgG aPL against domain I of ß2-glycoprotein I (ß2GPI) [anti-DI (aDI)] is associated with the pathogenesis of APS, an autoimmune disease defined by thrombosis and pregnancy morbidity. To date, however, no study has demonstrated direct pathogenicity of IgG aDI in vivo. In this proof-of-concept study, we designed a novel system to affinity purify polyclonal aDI aPL in order to assess its prothrombotic ability in a well-characterized mouse microcirculation model for APS. METHODS: Two polyclonal IgG fractions were isolated from serum of a patient with APS, both with high aPL activity but differing in aDI activity (aDI-rich and aDI-poor). These IgG fractions were tested for their pathogenic ability in an in vivo mouse model of thrombosis. Male CD1 mice were injected intraperitoneally with either aDI-rich or aDI-poor IgG; as a control, IgG isolated from healthy serum was used. A pinch injury was applied to the right femoral vein and thrombus dynamics and tissue factor activity in isolated tissue were evaluated. RESULTS: Both aDI-rich and aDI-poor IgG retained aCL and anti-ß2GPI activity, while only aDI-rich IgG displayed high aDI activity, as defined by our in-house cut-offs for positivity in each assay. aDI-rich IgG induced significantly larger thrombi in vivo compared with aDI-poor IgG (P < 0.0001). Similarly, aDI-rich IgG significantly enhanced the procoagulant activity of carotid artery endothelium and peritoneal macrophages isolated from experimental animals (P < 0.01). CONCLUSION: These data directly demonstrate that the ability to cause thrombosis in vivo is concentrated in the aDI fraction of aPL.

A novel pathway for human endothelial cell activation by antiphospholipid/anti-ß2 glycoprotein I antibodies.

Antiphospholipid Abs (APLAs) are associated with thrombosis and recurrent fetal loss. These Abs are primarily directed against phospholipid-binding proteins, particularly ß(2)GPI, and activate endothelial cells (ECs) in a ß(2)GPI-dependent manner after binding of ß(2)GPI to EC annexin A2. Because annexin A2 is not a transmembrane protein, the mechanisms of APLA/anti-ß(2)GPI Ab-mediated EC activation are uncertain, although a role for a TLR4/myeloid differentiation factor 88-dependent pathway leading to activation of NF-κB has been proposed. In the present study, we confirm a critical role for TLR4 in anti-ß(2)GPI Ab-mediated EC activation and demonstrate that signaling through TLR4 is mediated through the assembly of a multiprotein signaling complex on the EC surface that includes annexin A2, TLR4, calreticulin, and nucleolin. An essential role for each of these proteins in cell activation is suggested by the fact that inhibiting the expression of each using specific siRNAs blocked EC activation mediated by APLAs/anti-ß(2)GPI Abs. These results provide new evidence for novel protein-protein interactions on ECs that may contribute to EC activation and the pathogenesis of APLA/anti-ß(2)GPI-associated thrombosis and suggest potential new targets for therapeutic intervention in antiphospholipid syndrome.

Mitochondrial dysfunction in antiphospholipid syndrome: implications in the pathogenesis of the disease and effects of coenzyme Q(10) treatment.

The exact mechanisms underlying the role of oxidative stress in the pathogenesis and the prothrombotic or proinflammatory status of antiphospholipid syndrome (APS) remain unknown. Here, we investigate the role of oxidative stress and mitochondrial dysfunction in the proatherothrombotic status of APS patients induced by IgG-antiphospholipid antibodies and the beneficial effects of supplementing cells with coenzyme Q(10) (CoQ(10)). A significant increase in relevant prothrombotic and inflammatory parameters in 43 APS patients was found compared with 38 healthy donors. Increased peroxide production, nuclear abundance of Nrf2, antioxidant enzymatic activity, decreased intracellular glutathione, and altered mitochondrial membrane potential were found in monocytes and neutrophils from APS patients. Accelerated atherosclerosis in APS patients was found associated with their inflammatory or oxidative status. CoQ(10) preincubation of healthy monocytes before IgG-antiphospholipid antibody treatment decreased oxidative stress, the percentage of cells with altered mitochondrial membrane potential, and the induced expression of tissue factor, VEGF, and Flt1. In addition, CoQ(10) significantly improved the ultrastructural preservation of mitochondria and prevented IgG-APS-induced fission mediated by Drp-1 and Fis-1 proteins. In conclusion, the oxidative perturbation in APS patient leukocytes, which is directly related to an inflammatory and pro-atherothrombotic status, relies on alterations in mitochondrial dynamics and metabolism that may be prevented, reverted, or both by treatment with CoQ(10).

Apolipoprotein E receptor 2 is involved in the thrombotic complications in a murine model of the antiphospholipid syndrome.

Antiphospholipid (aPL)/anti-ß(2) glycoprotein I (anti-ß(2)GPI) antibodies stimulates tissue factor (TF) expression within vasculature and in blood cells, thereby leading to increased thrombosis. Several cellular receptors have been proposed to mediate these effects, but no convincing evidence for the involvement of a specific one has been provided. We investigated the role of Apolipoprotein E receptor 2 (ApoER2') on the pathogenic effects of a patient-derived polyclonal aPL IgG preparation (IgG-APS), a murine anti-ß(2)GPI monoclonal antibody (E7) and of a constructed dimeric ß(2)GPI I (dimer), which in vitro mimics ß(2)GPI-antibody immune complexes, using an animal model of thrombosis, and ApoER2-deficient (-/-) mice. In wild type mice, IgG-APS, E7 and the dimer increased thrombus formation, carotid artery TF activity as well as peritoneal macrophage TF activity/expression. Those pathogenic effects were significantly reduced in ApoER2 (-/-) mice. In addition, those effects induced by the IgG-APS, by E7 and by the dimer were inhibited by treatment of wild-type mice with soluble binding domain 1 of ApoER2 (sBD1). Altogether these data show that ApoER2 is involved in pathogenesis of antiphospholipids antibodies.

Phospholipid scramblase 1 expression is enhanced in patients with antiphospholipid syndrome.

OBJECTIVE: Thrombus formation is the key event of vascular manifestations in antiphospholipid syndrome (APS). Phosphatidylserine (PS) is normally sequestered in the inner leaflet of cell membranes. Externalization of PS occurs during cell activation and is essential for promoting blood coagulation and for the binding of antiphospholipid antibodies (aPL) to cells. One of the molecules involved in PS externalization is phospholipid scramblase 1 (PLSCR1). We evaluated PLSCR1 expression on monocytes from APS patients and analyzed the in vitro effect of monoclonal aPL on PLSCR1 expression. PATIENTS AND METHODS: Forty patients with APS were investigated. In vitro experiments were performed in monocyte cell lines incubated with monoclonal aPL. PLSCR1 expression was determined by quantitative real-time polymerase chain reactions. PS exposure on CD14(+) cell surface was analyzed by flow cytometry. RESULTS: Levels of full-length PLSCR1 messenger RNA (mRNA) were significantly increased in APS patients compared with healthy controls (2.4 ± 1.2 vs. 1.3 ± 0.4, respectively, p < 0.001). In cultured monocytes, interferon alpha enhanced tissue-factor expression mediated by ß2-glycoprotein-I-dependent monoclonal anticardiolipin antibody. CONCLUSIONS: Monocytes in APS patients had increased PLSCR1 mRNA expression.

Neutrophil activation by the tissue factor/Factor VIIa/PAR2 axis mediates fetal death in a mouse model of antiphospholipid syndrome.

Women with antiphospholipid syndrome (APS), a condition characterized by the presence of antiphospholipid antibodies (aPL), often suffer pregnancy-related complications, including miscarriage. We have previously shown that C5a induction of tissue factor (TF) expression in neutrophils contributes to respiratory burst, trophoblast injury, and pregnancy loss in mice treated with aPL. Here we analyzed how TF contributes to neutrophil activation and trophoblast injury in this model. Neutrophils from aPL-treated mice expressed protease-activated receptor 2 (PAR2), and stimulation of this receptor led to neutrophil activation, trophoblast injury, and fetal death. An antibody specific for human TF that has little impact on coagulation, but potently inhibits TF/Factor VIIa (FVIIa) signaling through PAR2, inhibited aPL-induced neutrophil activation in mice that expressed human TF. Genetic deletion of the TF cytoplasmic domain, which allows interaction between TF and PAR2, reduced aPL-induced neutrophil activation in aPL-treated mice. Par2-/- mice treated with aPL exhibited reduced neutrophil activation and normal pregnancies, which indicates that PAR2 plays an important role in the pathogenesis of aPL-induced fetal injury. We also demonstrated that simvastatin and pravastatin decreased TF and PAR2 expression on neutrophils and prevented pregnancy loss. Our results suggest that TF/FVIIa/PAR2 signaling mediates neutrophil activation and fetal death in APS and that statins may be a good treatment for women with aPL-induced pregnancy complications.

Annexin A2 is involved in antiphospholipid antibody-mediated pathogenic effects in vitro and in vivo.

Antiphospholipid (aPL) antibodies recognize receptor-bound beta(2) glycoprotein I (beta(2)GPI) on target cells, and induce an intracellular signaling and a procoagulant/proinflammatory phenotype that leads to thrombosis. Evidence indicates that annexin A2 (A2), a receptor for tissue plasminogen activator and plasminogen, binds beta(2)GPI on target cells. However, whether A2 mediates pathogenic effects of aPL antibodies in vivo is unknown. In this work, we studied the effects of human aPL antibodies in A2-deficient (A2(-/-)) mice. A2(-/-) and A2(+/+) mice were injected with immunoglobulin G (IgG) isolated from either a patient with antiphospholipid syndrome (IgG-APS), a healthy control subject (IgG-normal human serum), a monoclonal anti-beta(2)GPI antibody (4C5), an anti-A2 monoclonal antibody, or monoclonal antibody of irrelevant specificity as control. We found that, after IgG-APS or 4C5 injections and vascular injury, mean thrombus size was significantly smaller and tissue factor activity was significantly less in A2(-/-) mice compared with A2(+/+) mice. The expression of vascular cell adhesion molecule-1 induced by IgG-APS or 4C5 in explanted A2(-/-) aorta was also significantly reduced compared with A2(+/+) mice. Interestingly, anti-A2 monoclonal antibody significantly decreased aPL-induced expression of intercellular cell adhesion molecule-1, E-selectin, and tissue factor activity on cultured endothelial cells. Together, these data indicate for the first time that A2 mediates the pathogenic effects of aPL antibodies in vivo and in vitro APS.

Decreased expression of heparin-binding epidermal growth factor-like growth factor as a newly identified pathogenic mechanism of antiphospholipid-mediated defective placentation.

OBJECTIVE: Heparin-binding epidermal growth factor-like growth factor (HB-EGF) plays a role in blastocyst implantation and is down-regulated in preeclampsia and in hypertensive pregnancy disorders associated with defective extravillous trophoblast invasion. Defective placentation and severe preeclampsia are also features of the antiphospholipid syndrome (APS). The purpose of this study was to investigate whether abnormal HB-EGF expression plays a pathogenic role in antiphospholipid antibody (aPL)-mediated defective placentation. METHODS: HB-EGF expression in placental tissue was evaluated by Western blotting and messenger RNA analysis in normal and APS placentae. Polyclonal IgG fractions or monoclonal beta(2)-glycoprotein I-dependent aPL and their respective controls were investigated for the following 4 features: their binding to human trophoblast monolayers, as determined by cell enzyme-linked immunosorbent assay (ELISA); their effect on HB-EGF expression by Western blotting in trophoblast cell extracts as well as by ELISA as a protein secreted in the culture supernatants; their inhibitory effect on in vitro trophoblast invasiveness, as evaluated by Matrigel assay; and their inhibitory effect on matrix metalloproteinase (MMP) levels, as measured by gelatin zymography. Experiments were also performed in the presence of serial concentrations of heparin or recombinant HB-EGF. RESULTS: Placental APS tissue displayed reduced expression of HB-EGF. Polyclonal and monoclonal aPL bound to trophoblast monolayers and significantly reduced the in vitro synthesis and secretion of HB-EGF. Heparin inhibited aPL binding and restored HB-EGF expression in a dose-dependent manner. Addition of recombinant HB-EGF reduced the in vitro aPL-induced inhibition of Matrigel invasiveness as well as MMP-2 levels. CONCLUSION: These preliminary findings suggest that the reduction of aPL-mediated HB-EGF represents an additional mechanism that is responsible for the defective placentation associated with APS and that heparin protects from aPL-induced damage by inhibiting antibody binding.

Antiphospholipid antibodies affect human endometrial angiogenesis.

Antiphospholipid antibodies (aPL) represent an important risk factor for thrombosis and recurrent miscarriage in patients with antiphospholipid syndrome (APS). The mechanisms of aPL-mediated pregnancy failure have been researched. Previous studies demonstrated that aPL bind trophoblast cells, reducing proliferation, human chorionic gonadotrophin release, and in vitro invasiveness. Recent data suggest that aPL are also able to react with human decidual cells, inducing a proinflammatory phenotype. Decidua, a newly formed tissue on the maternal side of the human placenta, is characterized by active angiogenesis and structural modifications of the spiral arteries in early pregnancy. Since angiogenesis is a critical component of normal placentation, the purpose of our study was to evaluate the role of aPL on human endometrial angiogenesis. For this reason, we investigated the effect of aPL on in vitro endometrial endothelial cell (HEEC) angiogenesis, VEGF secretion by ELISA, matrix metalloproteinases (MMPs) activity by gelatin zymography, and DNA binding activity of NFKB by a sensitive multiwell colorimetric assay. Furthermore, we performed experiments to study whether aPL affects in vivo angiogenesis in a murine model. We found that aPL significantly decrease the number and the total length of the tubules formed by HEEC on in vitro Matrigel assay and reduce newly formed vessels in aPL-inoculated mice. Moreover, aPL reduce significantly both VEGF and MMPs production and, at the nuclear level, NFKB DNA binding activity. From our results, it appears that aPL are associated with an inhibition of angiogenesis, suggesting further additional mechanisms to explain the defective placentation in the APS.

Antiphospholipid Antibodies Are Not Associated with Clinical Parameters or Prognostic Outcomes in Patients with Non-Hodgkin's Lymphoma.

BACKGROUND: Antiphospholipid antibodies (APAs) are found quite frequently in patients with non-Hodgkin's lymphoma (NHL). However, the clinical significance of these antibodies is largely unknown. This study aims to delineate the clinical and prognostic role of APAs in NHL patients. PATIENTS AND METHODS: Consecutive patients of NHL were screened for lupus anticoagulant (LA), IgG/IgM anticardiolipin antibody, and IgG/IgM anti-ß2-glycoprotein I at the time of diagnosis. Baseline investigations, staging, and treatment were done as per institutional protocol. Patients were followed up until the last known outpatient visit or death. All were screened at each visit for any thromboembolic event. The association of APA status with baseline NHL characteristics and treatment response was evaluated by univariate analysis. Kaplan-Meier survival analysis was used to compare the final outcome in patients with or without APAs. Patients who were initially APA positive were retested for the corresponding antibody at the end of chemotherapy. RESULTS: Twenty-four out of 105 patients (22.8%) were APA positive at diagnosis. The presence of APA was not significantly associated with NHL stage, histology, International Prognostic Index score, activated partial thromboplastin time, or treatment response. The median duration of follow-up was 15 months. Only four patients developed venous thrombosis; none was APA positive. There was no statistically significant difference in overall survival between the two groups (p = 0.471). Patients, who were APA positive initially, tested negative at the end of treatment, irrespective of treatment response. CONCLUSION: APAs are encountered more frequently in NHL patients than in the general population. However, APAs do not correlate with disease severity, thrombosis risk, treatment outcome, or overall survival.

Effects of antibodies against human parvovirus B19 on angiogenic signaling.

Human parvovirus B19 (B19V) infection has symptoms similar to those of anti­phospholipid syndrome (APS). Antibodies against B19V­VP1 unique region (VP1u) exhibit activity similar to that of anti­phospholipid antibodies (aPLs) by inducing vascular endothelial cell adhesion factors and APS­like syndrome. Previous studies have identified an effect of aPLs on angiogenesis. However, little is understood regarding the effect of anti­B19V­VP1u antibodies on angiogenesis. The present study investigated the effects of anti­B19V­VP1u antibodies on the expression of adhesion molecules and angiogenic signaling using an aPL­induced human umbilical vein endothelial cell (HUVEC) model, and trypan blue staining and western blotting. The effect of B19V­VP1u antibodies on vascular endothelial growth factor (VEGF) expression in A549 cells, another well­known model used to study angiogenesis, was also examined. Significantly higher intracellular adhesion molecule 1 expression was observed following treatments with 10% fetal calf serum (FCS), aPL immunoglobulin G (IgG), B19V­VP1u IgG or B19V­NS1 IgG, compared with in the normal human (NH) IgG­treated cells. Conversely, significantly higher vascular cellular adhesion molecule 1 was only detected in HUVECs treated with B19V­VP1u IgG. Significantly increased integrin ß1 was detected in HUVECs treated with aPL IgG or B19V­VP1u IgG, whereas no difference in integrin ß1 was observed in those treated with 10% FCS, NH IgG or B19V­NS1 IgG. No difference in AKT­mTOR­S6 ribosomal protein (S6RP) signaling was observed in HUVECs treated with B19­VP1u IgG or B19V­NS1 IgG, compared with NH IgG­treated cells. Significantly higher human inducible factor­1α was detected in HUVECs treated with 10% FCS, aPL IgG, B19V­VP1u IgG or B19V­NS1 IgG, compared with in NH IgG­treated cells. However, there was no difference in the level of VEGF observed among HUVECs treated with NH IgG, B19V­VP1u IgG or B19V­NS1 IgG. Notably, no difference in VEGF level was observed in A549 cells treated with NH IgG, aPL IgG, B19V­VP1u IgG or B19V­NS1 IgG. These findings suggest that anti­B19V­VP1u antibodies may serve a role in activating adhesion molecules, but not in AKT­mTOR­S6RP signaling.

Tissue factor expression on monocytes induced by anti-phospholipid antibodies as a strong risk factor for thromboembolic complications in SLE patients.

Our aim was to clarify the role of anti-phospholipid antibodies in the pathogenesis of monocyte tissue factor (TF) expression and thromboembolic complications (TE) in patients with SLE. We examined cell surface expression of TF on monocytes in 93 SLE patients. Monocyte TF expression was significantly higher in SLE patients who had TE than in other SLE patients, and confirmed that the high expression of monocyte TF was a strong risk factor for TE. Furthermore, the presence of anti-cardiolipin/beta2-glycoprotein I antibodies (anti-CL/beta2-GPI) was strongly associated with the high expression of monocyte TF. We therefore studied the in vitro effect of IgG anti-CL/beta2-GPI on lipopolysaccharide (LPS)-induced expression of TF on monocytes in healthy peripheral blood and found that purified IgG containing anti-CL/beta2-GPI significantly enhanced LPS-induced monocyte TF expression. These results suggest that anti-CL/beta2-GPI cause persistently high TF expression on monocyte, which may contribute to the risk of thromboembolic events in SLE patients.

The effect of platelet activation on the hypercoagulability induced by murine monoclonal antiphospholipid antibodies.

BACKGROUND: To identify the mechanisms of the hypercoagulability associated with antiphospholipid antibodies, we investigated antibody-mediated platelet activation and interference of antibodies with phospholipid-dependent reactions. DESIGN AND METHODS: We used two murine monoclonal antibodies, one against beta(2)-glycoprotein I (7F6G), the other against prothrombin (28F4). Platelet activation was assessed by phospholipid-related platelet procoagulant activity. Endogenous thrombin potential without activated protein C (ETP(0)) and the activated protein C concentration that reduced the ETP(0) by 50% (IC(50)-APC) were determined by calibrated automated thrombography. RESULTS: Both monoclonal antibodies mimicked the effect of IgG in 11 out of a series of 40 patients with antiphospholipid antibodies in thrombography. In the presence of their target, 7F6G and 28F4 at 200 microg/mL exhibited comparatively low and high binding to platelets and elicited low and high levels of procoagulant phospholipids on platelet surface, respectively. In platelet-poor plasma, these antibodies induced a 1.6 and >12-fold increase in IC(50)-APC, respectively, thus providing evidence for a procoagulant effect independent of platelet activation. The 84% decrease in ETP(0) indicated that 28F4 also displayed an anticoagulant effect. In platelet-rich plasma, this anticoagulant effect was significantly less (23% decrease in ETP(0)), demonstrating that a high increase in procoagulant surfaces by platelet activation significantly antagonizes the anticoagulant effect of antiphospholipid antibodies. In both types of plasma, the inhibition of thrombin generation (reduced ETP(0)) was less than the inhibition of activated protein C activity (increased IC(50)-APC). CONCLUSIONS: Our findings show that platelet activation reinforces the hypercoagulability induced by competition between antiphospholipid antibodies/target complexes and pro- and anticoagulant complexes for phospholipid surfaces.

Antiphospholipid antibodies increase the levels of mitochondrial DNA in placental extracellular vesicles: Alarmin-g for preeclampsia.

The pathogenesis of preeclampsia remains unclear but placental factors are known to play a crucial role causing maternal endothelial cell dysfunction. One potential factor is placental micro- and nano- vesicles. Antiphospholipid antibodies (aPL) increase the risk of preeclampsia ten-fold, in part by damaging the mitochondria in the syncytiotrophoblast. Since mitochondrial DNA (mtDNA) is a danger- associated molecular pattern (DAMP/alarmin) that may activate endothelial cells, the aims of the current study were to investigate whether aPL affect the number of placental vesicles extruded, their mtDNA content and their ability to activate endothelial cells. Exposure of first trimester human placental explants to aPL affected neither the number nor size of extruded micro- and nano- vesicles (n = 5), however their levels of mtDNA were increased (n = 6). These vesicles significantly activated endothelial cells (n = 5), which was prevented by blocking toll-like receptor 9 (TLR-9), a receptor for extracellular DNA. Thus, aPL may increase the risk of preeclampsia in part by increasing the amount of mtDNA associated with placental vesicles. That mitochondrial DNA is recognised as a DAMP by TLR-9 to cause endothelial cell activation, raises the possibility that placental vesicles or TLR-9 might be a target for pharmaceutical intervention to reduce the consequences of aPL in pregnancy.

Thrombogenic effects of antiphospholipid antibodies are mediated by intercellular cell adhesion molecule-1, vascular cell adhesion molecule-1, and P-selectin.

Recent studies have shown that antiphospholipid (aPL) enhances expression of intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin on endothelial cells (ECs) and that these effects are correlated with increased adhesion of leukocytes to endothelium in cremaster muscle in vivo and with thrombosis in a mouse model. Activation of ECs by aPL may create a hypercoagulable state that precedes and contributes to thrombosis in patients with aPL syndrome (APS). This study proposed to examine whether this in vivo activation of ECs and enhanced thrombosis by aPL are mediated by ICAM-1, P-selectin, or VCAM-1. The dynamics of thrombus formation and the number of adhering leukocytes were studied in ICAM-1-deficient (ICAM-1(-/-)) mice or ICAM-1-/P-selectin-deficient (ICAM-1(-/-)/P-selectin(-/-)) mice treated with affinity-purified aPL antibodies (ap IgG-APS) or with control IgG and compared with wild-type mice treated in a similar fashion. In another set of experiments, the adhesion of leukocytes to cremaster muscle and the dynamics of thrombus formation were studied in CD1 mice treated with aPL or control IgG before and 30 minutes after intravenous infusion with 100 microg monoclonal antibody anti-VCAM-1. The results indicate that the enhanced adhesion of leukocytes to endothelium in wild-type mice was significantly reduced in ICAM-1(-/-) and completely abrogated in ICAM-1(-/-)/P-selectin(-/-) mice treated with ap IgG-APS compared with wild-type mice treated with ap IgG-APS (6.9+/-2.3, 0.4+/-0.4 versus 35+/-12, respectively). More importantly, this correlated with a significant reduction in thrombus size compared with wild-type mice treated with ap IgG-APS (895+/-259 microm(2), 859+/-243 microm(2) versus 3816+/-672 microm(2), respectively). Infusion of the mice with anti-VCAM-1 antibodies significantly reversed the enhanced adhesion of leukocytes (14.9+/-3 to 11.3+/-2.1) and thrombus size 3830+/-1008 microm(2) versus 876+/-548 microm(2)) in mice treated with ap IgG-APS. The data indicate that ICAM-1, P-selectin, and VCAM-1 expression are important in thrombotic complications by aPL antibodies and may provide novel targets for therapy in patients with APS.

Human extravillous trophoblasts bind but do not internalize antiphospholipid antibodies.

INTRODUCTION: Obstetric morbidity in women with antiphospholipid antibodies (aPLs) may reflect the adverse effects of aPLs on placental cells such as extravillous trophoblasts and the syncytiotrophoblast. Antiphospholipid antibodies may affect the syncytiotrophoblast after being internalised by members of the Low-density lipoprotein receptor (LDLR) family and the antigen of aPLs, ß2 glycoprotein I. AIM: This study aimed to determine whether aPL internalization was a mechanism by which aPLs adversely affect extravillous trophoblasts. METHOD: of STUDY: Fluorescently-labelled monoclonal aPLs IIC5 or ID2 were incubated with first trimester extravillous trophoblast outgrowths and visualized by microscopy. The subcellular expression of ß2 glycoprotein I and LDLR family members was investigated by live/permeabilised immunocytochemistry. RESULTS: Unlike the syncytiotrophoblast of anchoring villi, monoclonal aPLs were not internalised by extravillous trophoblasts, which expressed LDLR family members intracellularly. The aPL IIC5 bound to the surface of extravillous trophoblasts in a pattern similar to the extracellular expression of ß2 glycoprotein I. CONCLUSIONS: The mechanisms of action of aPLs are different in extravillous trophoblasts and the syncytiotrophoblast. The interaction of aPLs with the extravillous trophoblast surface, which may involve ß2 glycoprotein I, is consistent with reports that aPLs trigger intracellular signaling cascades through cell-surface receptors.

Antiphospholipid antibodies from antiphospholipid syndrome patients activate endothelial cells in vitro and in vivo.

BACKGROUND: Antiphospholipid (aPL) antibodies are associated with thrombosis in patients diagnosed with antiphospholipid syndrome (APS) and enhance thrombus formation in vivo in mice, but the mechanism of thrombosis by aPL is not completely understood. Although aPL antibodies have been shown to inhibit protein C activation and activate endothelial cells (ECs) in vitro, no study has examined whether these antibodies activate ECs in vivo. Therefore, human affinity-purified aPL (ap aPL) antibodies from APS patients were tested in a mouse model of microcirculation using the cremaster muscle that allows direct microscopic examination of thrombus formation and adhesion of white blood cells (WBCs) to ECs as an indication of EC activation in vivo. Adhesion molecule expression on human umbilical vein endothelial cells (HUVECs) after aPL exposure was performed to confirm EC activation in vitro. METHODS AND RESULTS: All 6 ap aPL antibodies significantly increased the expression of VCAM-1 (2.3- to 4.4-fold), with one of the antibodies also increasing the expression of E-selectin (1.6-fold) on HUVECs in vitro. In the in vivo experiments, each ap aPL antibody except for 1 preparation increased WBC sticking (mean number of WBCs ranged from 22.7 to 50.6) compared with control (14.4), which correlated with enhanced thrombus formation (mean thrombus size ranged from 1098 to 6476 versus 594 microm2 for control). CONCLUSIONS: Activation of ECs by aPL antibodies in vivo may create a prothrombotic state on ECs, which may be the first pathophysiological event of thrombosis in APS.

Fluvastatin increases the expression of adhesion molecules, monocyte chemoattractant protein-1 and tissue factor in HUVEC stimulated by patient IgG fractions containing antiphospholipid antibodies.

The presence of antiphospholipid antibodies (APLA) is associated with an increased risk of recurrent thrombosis and pregnancy loss. APLA are able to activate endothelial cells (EC) and induce an increase in the expression of inflammatory marker proteins, such as leukocyte adhesion molecules, tissue factor or the monocyte chemoattractant protein-1 (MCP-1). Our objective was to investigate the effect of statins on EC activation induced by APLA in vitro. IgG was purified from the plasma of six patients with APLA and from healthy controls. EC were incubated with patient IgG or with control IgG, in the presence or absence of 5microM of fluvastatin, and expression of the leukocyte adhesion molecules, VCAM-1 and E-selectin, analyzed by flow cytometry and by quantitative reverse transcriptase-PCR (QRT-PCR). The expression of tissue factor and the chemokine MCP-1 was analyzed by QRT-PCR alone. Incubation of EC with patient IgG increased the expression of VCAM-1, E-selectin, tissue factor and MCP-1. Prior treatment of the cells with fluvastatin further increased the expression of these proteins. The fluvastatin effect was reversed by co-incubation with mevalonate or geranylgeranylpyrophosphate and mimicked by the geranylgeranyl transferase inhibitor GGTI-286. Our results show that in cultured human EC, statins increase the extent of inflammatory activation induced by APLA. This effect appears to be mediated by an inhibitory effect of statins on one or more geranylgeranylated protein(s).