ABSTRACT
The antioxidant enzyme heme oxygenase (HO)-1, which catalyses the first and rate-limiting step of heme degradation, has major anti-inflammatory and immunomodulatory effects via its cell-type-specific functions in the endothelium. In the current study, we investigated whether the key endothelial adhesion and signalling receptor PECAM-1 (CD31) might be involved in the regulation of HO-1 gene expression in human endothelial cells (ECs). To this end PECAM-1 expression was down-regulated in human umbilical vein ECs (HUVECs) by an adenoviral vector-based knockdown approach. PECAM-1 knockdown markedly induced HO-1, but not the constitutive HO isoform HO-2. Nuclear translocation of the transcription factor NF-E2-related factor-2 (Nrf2), which is a master regulator of the inducible antioxidant cell response, and intracellular levels of reactive oxygen species (ROS) were increased in PECAM-1-deficient HUVECs, respectively. PECAM-1-dependent HO-1 regulation was also examined in PECAM-1 over-expressing Chinese hamster ovary and murine L-cells. Endogenous HO-1 gene expression and reporter gene activity of transiently transfected luciferase HO-1 promoter constructs with Nrf2 target sequences were decreased in PECAM-1 over-expressing cells. Moreover, a regulatory role of ROS for HO-1 regulation in these cells is demonstrated by studies with the antioxidant N-acetylcysteine and exogenous hydrogenperoxide. Finally, direct interaction of PECAM-1 with a native complex of its binding partner NB1 (CD177) and serine proteinase 3 (PR3) from human neutrophils, markedly induced HO-1 expression in HUVECs. Taken together, we demonstrate a functional link between HO-1 gene expression and PECAM-1 in human ECs, which might play a critical role in the regulation of inflammation.
Subject(s)
Endothelial Cells/metabolism , Heme Oxygenase-1/metabolism , NF-E2-Related Factor 2/metabolism , Platelet Endothelial Cell Adhesion Molecule-1/metabolism , Active Transport, Cell Nucleus , Animals , CHO Cells , Cell Line , Cricetinae , Cricetulus , GPI-Linked Proteins/metabolism , Gene Knockdown Techniques , Heme Oxygenase-1/genetics , Human Umbilical Vein Endothelial Cells , Humans , Isoantigens/metabolism , L Cells , Mice , Myeloblastin/metabolism , Platelet Endothelial Cell Adhesion Molecule-1/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reactive Oxygen Species/metabolism , Receptors, Cell Surface/metabolism , Signal Transduction , Up-RegulationABSTRACT
Alzheimer's disease (AD) is a neurodegenerative disorder primarily affecting regions of the brain responsible for higher cognitive functions. Immunization against ß-amyloid (Aß) in animal models of AD has been shown to be effective on the molecular level but also on the behavioral level. Recently, we reported naturally occurring autoantibodies against Aß (NAbs-Aß) being reduced in Alzheimer's disease patients. Here, we further investigated their physiological role: in epitope mapping studies, NAbs-Aß recognized the mid-/C-terminal end of Aß and preferentially bound to oligomers but failed to bind to monomers/fibrils. NAbs-Aß were able to interfere with Aß peptide toxicity, but NAbs-Aß did not readily clear senile plaques although early fleecy-like plaques were reduced. Administration of NAbs-Aß in transgenic mice improved the object location memory significantly, almost reaching performance levels of wild-type control mice. These findings suggest a novel physiological mechanism involving NAbs-Aß to dispose of proteins or peptides that are prone to forming toxic aggregates.
Subject(s)
Alzheimer Disease/genetics , Alzheimer Disease/immunology , Amyloid beta-Peptides/immunology , Autoantibodies/immunology , Alzheimer Disease/pathology , Alzheimer Disease/psychology , Animals , Animals, Genetically Modified , Antibody Formation , Behavior, Animal , Brain/pathology , Cells, Cultured , Chromatography, Gel , Disease Models, Animal , Epitopes , Female , Humans , Immunization , Immunoglobulin G/immunology , Immunohistochemistry , Mice , Mice, Inbred C57BL , Models, Molecular , Plaque, Amyloid/pathology , Surface Plasmon ResonanceABSTRACT
Intravenous immunoglobulins (IVIG) are currently used for therapeutic purposes in autoimmune disorders. Recently, we demonstrated the presence of naturally occurring antibodies against amyloid-beta (nAbs-Abeta) within the pool of IVIG. In this study, we compared different brands of IVIG for nAbs-Abeta and have found differences in the specificity of the nAbs-Abeta towards Abeta(1-40) and Abeta(1-42). We analyzed the influence of a pH-shift over the course of antibody storage using ELISA and investigated antibody dimerization at acidic and neutral pH as well as differences in the IgG subclass distributions among the IVIG using both HPLC and a nephelometric assay. Furthermore, we investigated the epitope region of purified nAbs-Abeta. The differences found in Abeta specificity are not directly proportionate to the binding nature of these antibodies when administered in vivo. This information, however, may serve as a guide when choosing the commercial source of IVIG for therapeutic applications in Alzheimer's disease.
Subject(s)
Amyloid beta-Peptides/immunology , Amyloid beta-Peptides/metabolism , Immunoglobulins, Intravenous/immunology , Autoantibodies/immunology , Autoantibodies/metabolism , Autoantibodies/therapeutic use , Binding, Competitive/drug effects , Binding, Competitive/immunology , Chromatography, Gel/methods , Cross Reactions/physiology , Enzyme-Linked Immunosorbent Assay/methods , Epitopes/immunology , Epitopes/metabolism , Humans , Immunoglobulins, Intravenous/therapeutic use , Nephelometry and Turbidimetry/methods , Peptide Fragments/immunologyABSTRACT
Aging is the most significant risk factor for Alzheimer disease (AD). The pathological hallmark of AD is the accumulation of aggregated amyloid-beta (Abeta) forms and insoluble plaques, mainly composed of Abeta, in the brain of the patient. Recently, we reported on the selection of D-enantiomeric, Abeta-binding peptides D1 and D3. D1 was selected against aggregated Abeta species to address diagnosis by in vivo imaging of amyloid plaques, whereas D3 was selected using low-molecular-weight Abeta species, therefore addressing therapeutical studies. Here, we use a surface plasmon resonance method to confirm that both peptides show the desired binding specificities.
Subject(s)
Amyloid beta-Peptides/metabolism , Oligopeptides/pharmacology , Peptide Fragments/pharmacology , Alzheimer Disease/diagnosis , Alzheimer Disease/metabolism , Alzheimer Disease/pathology , Amyloid/analysis , Amyloid/chemistry , Amyloid beta-Peptides/chemistry , Amyloid beta-Peptides/drug effects , Humans , Oligopeptides/chemistry , Oligopeptides/metabolism , Peptide Fragments/chemistry , Peptide Fragments/isolation & purification , Peptide Fragments/metabolism , Plaque, Amyloid/chemistry , Plaque, Amyloid/drug effects , Plaque, Amyloid/metabolism , Protein Binding , Protein Isoforms/analysis , Protein Isoforms/chemistry , Protein Multimerization/drug effects , Substrate Specificity , Surface Plasmon ResonanceABSTRACT
Factor VII-activating protease (FSAP) circulates as an inactive zymogen in the plasma. FSAP also regulates fibrinolysis by activating pro-urokinase or cellular activation via cleavage of platelet-derived growth factor BB (PDGF-BB). As the Marburg I polymorphism of FSAP, with reduced enzymatic activity, is a risk factor for atherosclerosis and liver fibrosis, the regulation of FSAP activity is of major importance. FSAP is activated by an auto-catalytic mechanism, which is amplified by heparin. To further investigate the structural requirements of polyanions for controlling FSAP activity, we performed binding, activation and inhibition studies using heparin and derivatives with altered size and charge, as well as other glycosaminoglycans. Heparin was effective in binding to and activating FSAP in a size- and charge density-dependent manner. Polyphosphate was more potent than heparin with regard to its interactions with FSAP. Heparin was also an effective co-factor for inhibition of FSAP by plasminogen activator inhibitor 1 (PAI-1) and antithrombin, whereas polyphosphate served as co-factor for the inhibition of FSAP by PAI-1 only. For FSAP-mediated inhibition of PDGF-BB-induced vascular smooth muscle cell proliferation, heparin as well as a polyphosphate served as efficient co-factors. Native mast cell-derived heparin exhibited identical properties to those of unfractionated heparin. Despite the strong effects of synthetic polyphosphate, the platelet-derived material was a weak activator of FSAP. Hence, negatively charged polymers with a high charge-to-size ratio are responsible for the activation of FSAP, and also act as co-factors for its inhibition by serine protease inhibitors.
