ALBERT

Description: Introduction: Antiphospholipid syndrome (APS) is an autoimmune disorder caused by "antiphospholipid" antibodies (aPL) directed against β2-glycoprotein I (β2GPI). In the presence of β2GPI, aPL activate endothelial cells through TLR4-mediated and other pathways. Previous studies suggest that a region encompassing amino acids 39-43 of β2GPI domain 1 comprises an important binding site for pathogenic aPL. However, due to difficulty in expressing full-length recombinant β2GPI, antibody specificity has only been tested using individual, recombinant β2GPI domains. Further definition of antibody specificity using intact recombinant β2GPI provides a new approach for diagnostic and mechanistic studies. In the present study we describe the expression of full-length, wild-type recombinant β2GPI and site-directed mutants encompassing the putative β2GPI binding region in domain 1. Binding of APS patient-derived anti-β2GPI antibodies to these proteins and their ability to support anti-β2GPI-mediated endothelial cell activation was determined. Methods: Full length APOH cDNA with a modified signal peptide (spm) was cloned into the lentiviral vector pLenti CMV Puro DEST. We generated two mutants spanning the 39 to 43 amino acid region: SEGVG (R39S; G40E; M42V; R43G) and AAGMA (R39A; G40A; R43A). The final recombinant plasmids were termed as pDEST-spmAPOH-WT; pDEST-spmAPOH-SEGVG and pDEST-spmAPOH-AAGMA. Lentivirus was produced using the Lentiviral Gateway Expression kit using GP2-293 (HEK) cells. Stable cell lines were generated by transducing HEK cells with APOH-lentivirus and selecting against puromycin. Stable cell lines were transferred to serum-free media and grown in suspension. Cell culture supernatants containing secreted β2GPI were filtered and concentrated, and recombinant β2GPI was purified using a hitrap-heparin column. Anti-β2GPI antibody binding to plasma-derived and rβ2GPI was determined using a standard β2GPI ELISA and SPR (Surface Plasmon Resonance) on a Biacore 3000. Biosensor analysis was performed by crosslinking β2GPI to carboxymethyl-dextran coated sensor chips using amine coupling. Anti-β2GPI antibodies at concentrations ranging from 1-15,000 nM, were flowed through channels until equilibrium binding was achieved, at which point dissociation was assessed over a 10 minute interval. The BIAevaluation program was used to calculate association and dissociation rates. Endothelial cell activation was assessed by measuring cell surface E-selectin expression after incubating cells with control IgG or anti-β2GPI antibodies in the presence or absence of wild-type or mutant rβ2GPI. Molecular modeling was performed using PyMol. Results: After transduction with lentivirus encoding β2GPI with a modified signal peptide, HEK-293 cells were able to express and efficiently secrete rβ2GPI and site-directed mutants. Using a standard β2GPI-ELISA and SPR we found no significant differences in binding affinity of aPL towards plasma-derived and wild-type rβ2GPI. However, in the anti-β2GPI ELISA, binding of patient-derived aPL (APS21) to both mutants was reduced by ~70% compared to wild-type rβ2GPI. Consistent with binding specificity for a region encompassing aa 39-43, the Kd for binding of APS21 anti-β2GPI antibodies to wild-type and rβ2GPI-SEGVG determined by SPR was 20 nM and 5000 nM, respectively. Moreover, the ability of patient-derived APS21 anti-β2GPI antibodies to activate endothelial cells was reduced by 〉60% in the presence of rβ2GPI-SEGVG compared to wild-type rβ2GPI. Molecular modeling of β2GPI demonstrated that a mutation in the aa 39-43 region is predicted to cause a change from a net positive to a net negative charge without any structural change (Figure 1). Conclusion: These studies are the first to assess binding of human anti-β2GPI antibodies to rβ2GPI and site-directed mutants expressed in mammalian cells. No significant differences between binding of aPL to plasma derived and wild-type rβ2GPI were observed. APS21 aPL has specificity towards epitope 39-43 of β2GPI domain 1 and its binding affinity to rβ2GPI-SEGVG was significantly reduced. Functional studies demonstrate the importance of β2GPI aa 39 to 43 in supporting endothelial cell activation by anti-β2GPI antibodies. These recombinant proteins should facilitate further studies concerning the role of aPL-β2GPI interactions in the diagnosis and pathogenesis of APS. Disclosures No relevant conflicts of interest to declare.

Description: Angiogenesis requires specific molecular regulation of complex vascular branching morphology. The cleaved form of high molecular weight kininogen (HKa) has been shown to cause apoptosis of proliferating endothelial cells, and inhibit angiogenesis in vivo. However, critical morphological effects of HKa on the complex branching morphology of angiogenic vascular trees have not been defined. We used the quail chorioallantoic membrane (CAM) angiogenesis assay, in which angiogenesis agonists or antagonists cause uniform perturbation of angiogenesis throughout the entire vascular tree, to assess the effects of HKa on branching morphology. HKa (1.56 to 50 micrograms) was applied uniformly to the CAM, and its effect on angiogenesis determined after 24 hours of incubation. VESGEN vascular analysis software, currently under development at NASA Glenn Research Center, was used to quantify major vessel parameters in low-magnification images of vascular trees automatically extracted by VESGEN. Results derived from qualitative observation and initial quantification of two experiments demonstrates that HKa significantly inhibits vascular growth. Vessel length density (Lv) and branch point density (Br) in CAM specimens treated with 25 micrograms of HKa decreased by 23% and 31% respectively (24 ± 1 cm/cm2 and 441 ± 43 /cm2, compared to 31 ± 0 cm/cm2 and 677 ± 51 /cm2 in vehicle-treated controls; mean +/− S.E. for n = 3). The fractal dimension (Df) of skeletonized vascular images is a highly sensitive indicator of vessel density that ranges from 1.35 for strong angiogenesis inhibition, to 1.40 and 1.48 for controls and strong angiogenesis stimulation, respectively. Df decreased to 1.35 ± 0.01 in the HKa-treated specimens, relative to 1.39 ± 0.00 in controls. However, thickening of large vessels in response to HKa resulted in a virtually equivalent vessel area density (Av) of 0.167 ± 0.012 cm2/cm2, relative to 0.174 ± 0.015 cm2/cm2 in controls. Further studies of the site-specific effects of HKa on branching morphology within the vascular tree are in progress. These results demonstrate that the antiangiogenic effects of HKa result in a unique antiangiogenic ’fingerprint’ vascular morphology in comparison to other angiogenesis inhibitors that include transforming growth factor-β1 and angiostatin.

Description: High molecular weight kininogen (HK) plays an important role in the assembly of the plasma kallikrein-kinin system. While the human genome contains a single copy of the kininogen gene, three copies exist in the rat (one encoding K-kininogen and two encoding T-kininogen). Here, we confirm that the mouse genome contains two homologous kininogen genes, mKng1 and mKng2. These genes are located on chromosome 16 in a head to head orientation with ∼ 30 kB intervening sequence, and are expressed in a tissue-specific manner. To determine the roles of these genes in murine development and physiology, we disrupted mKng1, which is expressed primarily in the liver. mKng1−/ − mice were viable, and immunoblotting using anti-bradykinin antibodies indicated a marked reduction in plasma HK and low molecular weight kininogen (LK), as well as ΔmHK-D5, a novel kininogen isoform that lacks kininogen domain 5. Clotting studies were also consistent with marked HK deficiency. Moreover, despite normal tail vein bleeding times, mKng1−/− mice displayed a significantly prolonged time to carotid artery occlusion following Rose Bengal administration and laser induced arterial injury. These results suggest that a single gene, mKng1, is responsible for production of plasma kininogen, and that plasma high molecular weight kininogen may contribute to induced arterial thrombosis in mice.

Description: The antiphospholipid antibody syndrome (APS) is characterized by the presence of circulating antiphospholipid antibodies (APLA) in association with thrombosis and/or recurrent fetal loss. Although initially thought to directly recognize anionic phospholipids, most APLA actually recognize phospholipid binding proteins, most commonly β2-glycoprotein I (ß2GPI). β2GPI binds with high affinity to annexin II on the surface of endothelial cells (Ma et al, JBC, 2000), and β2GPI-dependent “APLA” activate endothelial cells in a β2GPI-dependent manner (Simantov et al., JCI, 1995). Moreover, a preliminary report from our laboratory has demonstrated that cross-linking of annexin II bound β2GPI by APLA/anti-β2GPI antibodies leads to endothelial cell activation through a pathway involving NF-κB (Zhang et. al, Blood 2003). However, the mechanism by which annexin II cross-linking might induce signaling responses is uncertain, as annexin II is not a transmembrane protein. We thus have investigated the hypothesis that activation of endothelial cell signaling pathways by annexin II cross-linking might require a transmembrane “adaptor” protein that spans the plasma membrane, yet associates with cell surface annexin II. First, human umbilical vein endothelial cell (HUVEC) surface proteins were biotin labeled using the membrane impermeable biotinylation reagent NHS-LC-biotin, and labeled annexin II binding proteins were affinity purified on immobilized annexin II. Purified annexin II binding proteins were detected following 10% SDS-PAGE, transfer to PVDF and development of the membranes using streptavidinperoxidase and chemiluminescence. These studies revealed bands of ~83, ~79, ~62 and ~34 kD. None of these bands were affinity-purified on immobilized bovine serum albumin, suggesting that specific annexin II binding proteins were present on the surface of endothelial cells. To identify these proteins, affinity-purification of annexin II binding proteins from ~40 x 106 HUVEC was undertaken using Affi-Gel HZ to which 10 mg of recombinant annexin II was coupled. Elution of bound proteins from this column followed by SDS-PAGE and staining with Coomassie brilliant blue revealed proteins of similar Mr as those identified using cell-surface labeled HUVEC. These proteins were excised from the gel, and analyzed by LC-MS following in gel tryptic digestion. Results of these studies revealed that the ~83 kD band was the Toll-like receptor 4 (TLR-4), the ~79 kD band was nucleolin, the ~62 kD band was calreticulin, and the ~34 kD band was annexin II. While we have not yet analyzed the role of each of these proteins in APLA/anti-β2GPI antibody-mediated endothelial cell activation, we have performed preliminary studies to address the potential involvement of TLR4, as a recent report demonstrated potential involvement of MyD88, a downstream mediator of TLR4-dependent signaling, in the activation of endothelial cell lines by APLA/anti-β2GPI antibodies (Raschi et al., Blood 2003). Preliminary studies suggest that TLR4 co-immunoprecipitates with annexin II, and that APLA/anti-β2GPI antibody induced endothelial cell activation, but not that caused by TNF-α, was partially blocked following transfection of endothelial cells with TLR4 siRNA. In conclusion, these studies confirm that cross-linking of endothelial annexin II initiates APLA/anti-β2GPI antibody-induced endothelial cell activation, and suggests the involvement of TLR4, and perhaps other endothelial cell surface proteins.

Description: Introduction The association between malignancy and venous thromboembolism (VTE) is well established. Less is known about the independent impact of VTE, both symptomatic and incidental, on survival in patients with prostate cancer. Methods We conducted a retrospective cohort study of 457 consecutive patients with prostate cancer who received outpatient clinical care at the Cleveland Clinic from January 2006 to June 2006. Data were collected regarding clinical characteristics, treatment and outcomes. Fisher exact (for categorical variables) and t-test (continuous variables) were utilized to test associations with VTE and mortality. Survival functions were estimated using the Kaplan Meier method and a Cox regression model was used to model the mortality hazard ratio (HR) with date of diagnosis as time origin. Results The mean age of our cohort of patients was 65.86 ± 8.64 years. Three hundred and fifty eight (78.3%) had clinically localized disease, 76 (16.7%) had locally advanced disease and 41 (8.9%) had metastatic cancer at diagnosis. One hundred twenty four (27.1%) men underwent surgery, 315 (68.9%) received radiotherapy, 201 (44%) received hormonal therapy and 71 (15.5%) received chemotherapy. Complete follow up was available on 403 patients of which 109 (27%) died during the period of follow up. VTE occurred in 42 (9.2%) patients (33 deep vein thrombosis, 5 pulmonary embolism, and 4 patients with both DVT and PE), of which 27 (64.3%) were symptomatic and 15 (35.7%) were incidentally diagnosed. Twenty-three were outpatients and 17 were hospitalized when VTE was diagnosed. Metastatic disease (p

Description: Abstract 4315 Antiphospholipid syndrome (APS) is characterized by thrombosis and/or pregnancy loss in the presence of antiphospholipid antibodies (APLA). These antibodies are directed primarily against phospholipid-bound β2-glycoprotein I (β2GPI). Anti-β2GPI antibodies activate endothelial cells, enhancing the expression of adhesion molecules and tissue factor, and the secretion of proinflammatory cytokines. Krüppel-like factors (KLF) regulate endothelial cell inflammatory responses. KLF2 and KLF4 mediate anti-atherosclerotic and anti-inflammatory effects in endothelial cells, and we have hypothesized that alterations in the expression or activity of KLF2 or KLF4 may modulate the endothelial cell response to APLA. In preliminary studies, we have observed that endothelial cell activation induced by APLA/anti-β2GPI antibodies inhibits the expression of KLF2 and KLF4, and as demonstrated by our laboratory and others, is accompanied by activation of NF-kB. However, forced expression of KLF2 or KLF4 by plasmid-mediated transfection of endothelial cells inhibits neither the phosphorylation of ser536 of the p65 subunit of NF-kB, nor the nuclear translocation of p65 in response to APLA/anti-β2GPI antibodies. Despite the lack of effect on forced KLF2 or KLF4 expression in endothelial cells on p65 phosphorylation, expression of either of these factors inhibits NF-κB transcriptional activity with corresponding inhibition of cellular activation as measured by inhibition of cell-surface E-selectin expression as well as E-selectin promoter activity. Inhibition of NF-kB transcriptional activity by KLF2 and KLF4 appears to be due to recruitment of the CBP/p300 cofactor away from NF-kB by KLF2 or KLF4, since augmenting the cellular pool of CBP/p300 by transfection restores NF-κB activity and endothelial cell activation responses. Similarly, treatment of APLA-activated endothelial cells with CBP/p300 siRNA inhibits NF-kB transcriptional activity regardless of the levels of KLF2 or KLF4. These data suggest that APLA inhibit KLF expression and that these changes promote the acquisition of a prothrombotic endothelial cell phenotype. CBP/p300 may serve as a molecular switch that determines the relative antithrombotic activities of KLFs versus the prothrombotic, inflammatory responses induced by NF-kB in APLA/anti-β2GPI antibody activated endothelial cells. Disclosures: No relevant conflicts of interest to declare.