betadex and Necrosis

Antiphospholipid antibodies (aPL) are the strongest maternal risk factor for pre-eclampsia, a hypertensive disease of human pregnancy. Pre-eclampsia is triggered by a toxic factor released from the placenta that activates the maternal endothelium. Antiphospholipid antibodies cause the release of necrotic trophoblast debris from the placental syncytiotrophoblast and this debris can activate endothelial cells. In this study, we investigated how aPL affects syncytiotrophoblast death and production of necrotic trophoblast debris by examining the interaction between aPL and human first trimester placental explants. Human polyclonal and murine monoclonal aPL, but not control antibodies, were rapidly internalised by the syncytiotrophoblast. Inhibitors of endocytosis or the low-density lipoprotein receptor (LDLR) family, but not toll-like receptors, decreased the internalisation of aPL and prevented the release of necrotic trophoblast debris from the syncytiotrophoblast. Once internalised, aPL increased inner mitochondrial membrane leak and Cytochrome c release while depressing oxidative flux through Complex IV of the electron transport system in syncytiotrophoblast mitochondria. These data suggest that the human syncytiotrophoblast internalises aPL by antigen-dependent endocytosis involving LDLR family members. Once internalised by the syncytiotrophoblast, aPL affects the death-regulating mitochondria, causing extrusion of necrotic trophoblast debris which can activate maternal endothelial cells thereby contributing to the pathogenesis of pre-eclampsia.

Topics: Antibodies, Antiphospholipid; Antibodies, Monoclonal; Apoptosis; beta-Cyclodextrins; Cells, Cultured; Chloroquine; Cytochromes c; Electron Transport Complex IV; Endocytosis; Endothelial Cells; Female; Humans; Mitochondrial Membranes; Necrosis; Nitrobenzoates; Organ Culture Techniques; Placenta; Pre-Eclampsia; Pregnancy; Protein Transport; Receptors, LDL; Trophoblasts

Cyclodextrins (CDs) have been shown to improve physicochemical and biopharmaceutical properties of drugs when low solubility and low safety limit their use in the pharmaceutical field. Recently, we have developed new multi-substituted-β-CDs, hydroxypropyl-sulfobutyl ether-β-cyclodextrin (HPn-SBEm-β-CD). HPn-SBEm-β-CD exhibit low hemolysis, good solubility, strong inclusion ability, and an appropriate average molecular weight. In this study, we chose two products of HPn-SBEm-β-CD (HP(3)-SBE(2)-β-CD and HP(2)-SBE(3)-β-CD) and compared their effects to sulfobutyl ether-β-cyclodextrin (SBE(7)-β-CD), methyl-β-cyclodextrin (M-β-CD) and 2,6-di-O-methyl-β-cyclodextrin (DM-β-CD). We evaluated viability, membrane damage, induction of apoptosis and necrosis, cholesterol depletion, and morphological changes in human embryonic kidney 293A cells (HEK293A) in vitro. CDs caused a reduction of cell viability and increased LDH levels in a concentration-dependent manner. The effect of HP(3)-SBE(2)-β-CD or HP(2)-SBE(3)-β-CD on cell viability, membrane damage, and the induction of apoptosis and necrosis resembled that of SBE(7)-β-CD, whereas the effects were significantly lower for M-β-CD or DM-β-CD. HP(3)-SBE(2)-β-CD and HP(2)-SBE(3)-β-CD exhibited morphological changes at high concentrations. In conclusion, the results showed that cholesterol depletion may be as a marker for evaluating the cytotoxicity of novel β-CD derivatives. These results will provide useful information for HPn-SBEm-β-CD as a promising safe adjuvant for intravenous administration in the future.

Topics: Apoptosis; beta-Cyclodextrins; Biomarkers; Cell Membrane; Cell Nucleus; Cell Survival; Cholesterol; Flow Cytometry; HEK293 Cells; Humans; Kidney; Kidney Diseases; L-Lactate Dehydrogenase; Necrosis

BACKGROUND AND OBJECTIVE. Alzheimer's disease is a progressive neurodegenerative disease that is biochemically characterized by the accumulation of amyloid beta (Aβ) peptides in the brain. The current hypothesis suggests that Aβ oligomers rather than fibrillar aggregates are the most toxic species of Aβ though the mechanisms of their neurotoxicity are unclear. The authors have previously shown that small Aβ(1-42) oligomers at around 1 µM concentration caused rapid (in 24 h) neuronal death in cerebellar granule cell (CGC) cultures. In this study, we aimed to investigate whether protracted (up to 7 days) incubation of CGC cultures with lower submicromolar concentration of various aggregates of Aβ(1-42) had an effect on viability of neurons. In order to get some insight into the mechanism of Aβ-induced cell death, we also sought to determine whether extracellular Ca(2+) and process of endocytosis contributed to Aβ oligomer-induced neurotoxicity and whether pharmacological interventions into these processes would prevent Aβ oligomer-induced cell death. MATERIAL AND METHODS. Primary cultures of CGC were treated with various aggregate forms of Aβ(1-42). Cell viability was assessed by fluorescent microscopy using propidium iodide and Hoechst 33342 staining. RESULTS. Exposure of neurons to 500 nM Aβ(1-42) oligomers for 72-168 h caused extensive neuronal necrosis. Lower concentrations (100-250 nM) were not toxic to cells during 7 days of incubation. Aβ(1-42) monomers and fibrils had no effect on neuronal viability even after 7 days of incubation. Treatment of neurons with EGTA, steroid hormone 17β-estradiol, and methyl-β-cyclodextrin significantly reduced Aβ(1-42) oligomers-induced neuronal death. CONCLUSIONS. The results show that submicromolar concentrations of Aβ(1-42) oligomers were highly toxic to neurons during protracted incubation inducing neuronal necrosis that can be prevented by chelating extracellular Ca(2+) with EGTA, inhibiting endocytosis with methyl-β-cyclodextrin, or by estradiol, which may protect against mitochondrial permeability transition pore opening.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Apoptosis; beta-Cyclodextrins; Cells, Cultured; Cerebellum; Egtazic Acid; Endocytosis; Estradiol; Microscopy, Fluorescence; Necrosis; Neurons; Propidium; Rats; Rats, Wistar

To study the effect of anandamide (AEA) on necrosis in HepG2 cells and to explore the role of AEA in progression of liver cancer.. Localization of the fatty acid hydrolytic enzyme (FAAH), cannabinoid receptors 1(CB1) and cannabinoid receptors 2 (CB2) proteins was detected in L02 and HepG2 cells using immunofluorescence. L02 and HepG2 cells were treated with different concentrations of AEA and methyl-beta-cyclodextrin, and the rates of cells necrosis were examined by PI stain. Meanwhile, the expression levels of FAAH, CB1 and CB2 receptor proteins, as well as P38 mitogen-activated protein kinase (p-P38 MAPK) and c-Jun-NH2-terminal kinase (p-JNK) proteins, were analyzed by Western blot.. The FAAH, CB1 and CB2 receptor proteins were observed both in cytoplasm and on membrane in L02 and HepG2 cells. The expression level of FAAH protein was higher in HepG2 than in L02 cells. The expression level of CB1 receptor protein was very low in both L02 and HepG2 cells. The expression level of CB2 receptor protein was high in both L02 and HepG2 cells. AEA treatment induced necrosis in HepG2 cells but not in L02 cells. Methyl-beta-cyclodextrin treatment prevented necrosis in HepG2 cells (t = 3.702; 5.274; 3.503, P less than 0.05). The expression patterns of FAAH, CB1 and CB2 receptor protein in L02 and HepG2 cells were confirmed by western blot, which were consistent with the immunofluorescence results. AEA treatment increased the levels of p-P38MAPK and p-JNK proteins in a dose-dependent manner in HepG2 cells (F = 11.908; 26.054, P less than 0.05) and the increase can be partially by prevented by MCD (t = 2.801; t = 12.829, P less than 0.05).. AEA treatment induces necrosis in HepG2 cells via CB1 and CB2 receptors and lipid rafts.

Topics: Amidohydrolases; Arachidonic Acids; beta-Cyclodextrins; Cannabinoid Receptor Modulators; Cholesterol; Endocannabinoids; Hep G2 Cells; Humans; JNK Mitogen-Activated Protein Kinases; Necrosis; p38 Mitogen-Activated Protein Kinases; Polyunsaturated Alkamides; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Signal Transduction