After 72 hours of culture, SCTs were obtained following spontaneous differentiation of cytotrophoblasts and were used for the studies described below. Incubation with Antiphospholipid Antibodies and Hydroxychloroquine To determine the effects of aPL N-(p-Coumaroyl) Serotonin antibodies on AnxA5 and whether HCQ might alter the effect as previously described in other systems,15 aPL or control antibodies (polyclonal antibody at 0.2 mg/ml and mAb at 0.1 mg/ml), together with either HCQ (1 g/ml in HBS) or buffer control (HBS) in the DMEM/F12/FBS medium were added to the SCTSs and incubated in humidified atmosphere for 24 hours. The placental anticoagulant protein annexin A5 (AnxA5) is highly expressed by syncytiotrophoblasts (SCTs) in an apparently constitutive manner.1 The potent anticoagulant properties of N-(p-Coumaroyl) Serotonin AnxA5 result from its forming 2-dimensional crystals over anionic phospholipids2 that shield them from availability for serving as cofactors for coagulation enzyme reactions.3 AnxA5 localizes on apical membranes of placental SCTs,1 an optimal anatomic position for the protein to play a thrombomodulatory role in maintaining the fluidity of intervillous blood circulation. Evidence from animal studies supports this concept; pregnant mice infused with anti-AnxA5 antibodies developed placental necrosis and fibrosis along with fetal resorption. 4 There is also evidence for such a role in humans, although it is less direct because of ethical concerns that limit such experimentation. Patients with preeclampsia and fetal growth restriction had reduced expression of placental AnxA5 compared to matched controls.5 Women with histories for unexplained recurrent spontaneous pregnancy losses have reduced AnxA5 levels and resistance to the anticoagulant activity of AnxA5.6 A common haplotype in the promoter region of the AnxA5 gene C designated M2 C was associated with reduced placental expression of AnxA57,8 and with increased risk for recurrent spontaneous pregnancy losses9,10 The antiphospholipid (aPL) syndrome (APS) is an Mouse monoclonal to CK4. Reacts exclusively with cytokeratin 4 which is present in noncornifying squamous epithelium, including cornea and transitional epithelium. Cells in certain ciliated pseudostratified epithelia and ductal epithelia of various exocrine glands are also positive. Normally keratin 4 is not present in the layers of the epidermis, but should be detectable in glandular tissue of the skin ,sweat glands). Skin epidermis contains mainly cytokeratins 14 and 19 ,in the basal layer) and cytokeratin 1 and 10 in the cornifying layers. Cytokeratin 4 has a molecular weight of approximately 59 kDa. acquired autoimmune thrombophilic condition that is a cause of pregnancy complications attributable to placental insufficiency including: recurrent pregnancy losses and other including IUGR, oligohydramnios, preeclampsia/toxemia and placental abruption.11 aPL antibodies reduced the levels of AnxA5 on placental villous SCTs,12 cultured BeWo trophoblasts,13C15 and primary cultures of SCTs,14 and reduce the anticoagulant activity of AnxA5 on the cells.14,15 The aPL-mediated reduction of AnxA5 has been confirmed to be due to competitive displacement of the protein by several different methods N-(p-Coumaroyl) Serotonin including atomic force microscopy,16 ellipsometry,17 microtiter plate assays,17,18 measurements of AnxA5 binding to phospholipid suspensions,17 flow cytometry,19,20, and fluorescence imaging.21 We were motivated to investigate whether hydroxychloroquine (HCQ) might directly affect the aPL-AnxA5 thrombogenic mechanism because of the drugs interesting chemical structure and because it reduced thrombosis in an animal model of APS.22 Observational studies in humans have also suggested a beneficial effect for the drug in reducing the risk of thrombosis23C28 We showed, through ellipsometry and atomic force microscopic imaging of aPL immune complexes on planar phospholipid bilayers, that HCQ directly disrupts the formation of aPL immune complexes15, 29 and that this restores AnxA5 binding and crystallization on the planar bilayers,15,29 Also, using quantitative immunoassays, we demonstrated that the drug also reduced aPL binding and restored AnxA5 expression on cultured BeWo trophoblasts.15 Since those results were obtained through immunoassay measurements on a choriocarcinoma-derived trophoblast model and did not provide information on the localization of the proteins, we thought it critical to image primary cultures of human syncytiotophoblasts (SCTs) to study the effects HCQ on the distribution of antibodies and AnxA5. Materials and Methods Reagents The research protocol was approved by the institutional review board of Montefiore Medical Center, which granted permission for the use of excess plasmas from APS patients that had been obtained from clinical assays or plasmapheresate discards, and were anonymized. Human polyclonal antibody immunoglobulin G (IgG) fractions were isolated from citrated plasma of patient with severe APS and a normal control subject with a protein G column, as described by Sammaritano et al.30 The patient had severe primary APS, manifested by recurrent spontaneous pregnancy losses, deep vein thrombosis, pulmonary embolism, stroke and high titers of anticardiolipin (aCL) IgG (25.3C30.6 GPL) and antiphosphatidylserine IgG (78.0C92.5 GPS), and positive lupus anticoagulant tests by standard dilute Russell viper venom time assays performed with mixing and confirmatory steps. The preparation of aPL antibodies from the patient was compared to IgG isolated from control plasma. The findings were validated with a previously characterized human aPL monoclonal antibody (mAb) IgG, designated IS4 that was generated from a cell line generously provided by Dr. Pojen P. Chen (Department of Medicine, Division.

After 72 hours of culture, SCTs were obtained following spontaneous differentiation of cytotrophoblasts and were used for the studies described below