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      Molecular Mechanism for Cellular Response to β-Escin and Its Therapeutic Implications

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          Abstract

          β-escin is a mixture of triterpene saponins isolated from the horse chestnut seeds ( Aesculus hippocastanum L.). The anti-edematous, anti-inflammatory and venotonic properties of β-escin have been the most extensively clinically investigated effects of this plant-based drug and randomized controlled trials have proved the efficacy of β-escin for the treatment of chronic venous insufficiency. However, despite the clinical recognition of the drug its pharmacological mechanism of action still remains largely elusive. To determine the cellular and molecular basis for the therapeutic effectiveness of β-escin we performed discovery and targeted proteomic analyses and in vitro evaluation of cellular and molecular responses in human endothelial cells under inflammatory conditions. Our results demonstrate that in endothelial cells β-escin potently induces cholesterol synthesis which is rapidly followed with marked fall in actin cytoskeleton integrity. The concomitant changes in cell functioning result in a significantly diminished responses to TNF-α stimulation. These include reduced migration, alleviated endothelial monolayer permeability, and inhibition of NFκB signal transduction leading to down-expression of TNF-α—induced effector proteins. Moreover, the study provides evidence for novel therapeutic potential of β-escin beyond the current vascular indications.

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          Most cited references45

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          The biological action of saponins in animal systems: a review.

          Saponins are steroid or triterpenoid glycosides, common in a large number of plants and plant products that are important in human and animal nutrition. Several biological effects have been ascribed to saponins. Extensive research has been carried out into the membrane-permeabilising, immunostimulant, hypocholesterolaemic and anticarcinogenic properties of saponins and they have also been found to significantly affect growth, feed intake and reproduction in animals. These structurally diverse compounds have also been observed to kill protozoans and molluscs, to be antioxidants, to impair the digestion of protein and the uptake of vitamins and minerals in the gut, to cause hypoglycaemia, and to act as antifungal and antiviral agents. These compounds can thus affect animals in a host of different ways both positive and negative.
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            The amyloid precursor protein has a flexible transmembrane domain and binds cholesterol.

            C99 is the transmembrane carboxyl-terminal domain of the amyloid precursor protein that is cleaved by γ-secretase to release the amyloid-β polypeptides, which are associated with Alzheimer's disease. Nuclear magnetic resonance and electron paramagnetic resonance spectroscopy show that the extracellular amino terminus of C99 includes a surface-embedded "N-helix" followed by a short "N-loop" connecting to the transmembrane domain (TMD). The TMD is a flexibly curved α helix, making it well suited for processive cleavage by γ-secretase. Titration of C99 reveals a binding site for cholesterol, providing mechanistic insight into how cholesterol promotes amyloidogenesis. Membrane-buried GXXXG motifs (G, Gly; X, any amino acid), which have an established role in oligomerization, were also shown to play a key role in cholesterol binding. The structure and cholesterol binding properties of C99 may aid in the design of Alzheimer's therapeutics.
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              The contribution of activated astrocytes to Aβ production: Implications for Alzheimer's disease pathogenesis

              Background β-Amyloid (Aβ) plays a central role in Alzheimer's disease (AD) pathogenesis. Neurons are major sources of Aβ in the brain. However, astrocytes outnumber neurons by at least five-fold. Thus, even a small level of astrocytic Aβ production could make a significant contribution to Aβ burden in AD. Moreover, activated astrocytes may increase Aβ generation. β-Site APP cleaving enzyme 1 (BACE1) cleavage of amyloid precursor protein (APP) initiates Aβ production. Here, we explored whether pro-inflammatory cytokines or Aβ42 would increase astrocytic levels of BACE1, APP, and β-secretase processing, implying a feed-forward mechanism of astrocytic Aβ production. Methods Mouse primary astrocytes were treated with combinations of LPS, TNF-α, IFN-γ, and IL-1β and analyzed by immunoblot and ELISA for endogenous BACE1, APP, and secreted Aβ40 levels. Inhibition of JAK and iNOS signaling in TNF-α+IFN-γ-stimulated astrocytes was also analyzed. In addition, C57BL/6J or Tg2576 mouse astrocytes were treated with oligomeric or fibrillar Aβ42 and analyzed by immunoblot for levels of BACE1, APP, and APPsβsw. Astrocytic BACE1 and APP mRNA levels were measured by TaqMan RT-PCR. Results TNF-α+IFN-γ stimulation significantly increased levels of astrocytic BACE1, APP, and secreted Aβ40. BACE1 and APP elevations were post-transcriptional at early time-points, but became transcriptional with longer TNF-α+IFN-γ treatment. Despite a ~4-fold increase in astrocytic BACE1 protein level following TNF-α+IFN-γ stimulation, BACE1 mRNA level was significantly decreased suggesting a post-transcriptional mechanism. Inhibition of iNOS and JAK did not reduce TNF-α+IFN-γ-stimulated elevation of astrocytic BACE1, APP, and Aβ40, except that JAK inhibition blocked the APP increase. Finally, oligomeric and fibrillar Aβ42 dramatically increased levels of astrocytic BACE1, APP, and APPsβsw through transcriptional mechanisms, at least in part. Conclusions Cytokines including TNF-α+IFN-γ increase levels of endogenous BACE1, APP, and Aβ and stimulate amyloidogenic APP processing in astrocytes. Oligomeric and fibrillar Aβ42 also increase levels of astrocytic BACE1, APP, and β-secretase processing. Together, our results suggest a cytokine- and Aβ42-driven feed-forward mechanism that promotes astrocytic Aβ production. Given that astrocytes greatly outnumber neurons, activated astrocytes may represent significant sources of Aβ during neuroinflammation in AD.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                11 October 2016
                2016
                : 11
                : 10
                : e0164365
                Affiliations
                [1 ]Laboratory of Mass Spectrometry, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland
                [2 ]Centre for Preclinical Research and Technology, Department of Immunology, Biochemistry and Nutrition, Medical University of Warsaw, Warsaw, Poland
                [3 ]Department of Molecular and Translational Oncology, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Warsaw, Poland
                [4 ]Department of Cytology, Faculty of Biology, University of Warsaw, Warsaw, Miecznikowa 1, 02–096 Warsaw, Poland
                [5 ]Laboratory for Microarray Analysis CORELAB, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland
                University of Sheffield, UNITED KINGDOM
                Author notes

                Competing Interests: DD, OZ-S, DM, MK and KK have a potential financial competing interest related to a patent application EP15001035.3 “New therapy for Alzheimer’s disease. OZ-S, IG, DM, MK and KK have a potential financial competing interest related to a patent application PCT/IB2016/000187 “Protoescigenin derivative, process of its preparation, use of said compound and pharmaceutical composition comprising that compound. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

                • Conceptualization: DD OZ-S MK IG DM AF MDa KK.

                • Data curation: DD AP MK IG AF KK.

                • Formal analysis: MK KK.

                • Funding acquisition: DD KK.

                • Investigation: DD OZ-S AP MDu MK IG DM AF MDa KK.

                • Methodology: DD OZ-S AP MDu MK IG DM AF MDa KK.

                • Project administration: DM MK KK.

                • Supervision: MK MDa KK.

                • Validation: DD AP OZ-S IG MDu AF MDa KK.

                • Visualization: IG MK KK.

                • Writing – original draft: DD OZ-S AP MDu MK IG DM AF MDa KK.

                • Writing – review & editing: MK KK.

                Article
                PONE-D-16-06656
                10.1371/journal.pone.0164365
                5058498
                27727329
                e983ec5f-524c-4760-b973-dcff7e7cd331
                © 2016 Domanski et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 16 February 2016
                : 23 September 2016
                Page count
                Figures: 7, Tables: 1, Pages: 18
                Funding
                Funded by: European Union Operational Programme “Innovative Economy”
                Award ID: POIG.01.01.02-14-072/09
                Award Recipient :
                Funded by: Foundation for Polish Science
                Award ID: HOMING PLUS/2011-3/1
                Award Recipient :
                Funded by: Foundation for Polish Science
                Award ID: Pomost/2013-8/8
                Award Recipient :
                This work was funded by the European Union under the Operational Programme “Innovative Economy” grant no. POIG.01.01.02-14-072/09 entitled “Search of innovative endothelial drug in a group of new escin derivatives” (KK). Funding was also provided by the Homing Plus for the project HOMING PLUS/2011-3/1 “Development of targeted mass spectrometry-based proteomic assays for the discovery of a novel endothelium-specific pharmaceutical for the treatment of human vascular diseases” from the Foundation for Polish Science (FNP) co-financed from EU structural funds within Action 1.2 “Strengthening the personnel potential of science” POIG 2007-2013 of the Innovative Economy Operational Programme (DD). Foundation for Polish Science supported the project by providing funds for the project Bridge/2013-8/8 “ß-escin—a new agent in the skeletal muscle regeneration improvement?” (KK). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. All the below mentioned funders provided support in form of salaries, but also all the materials for the study were purchased with the obtained funding: 1. European Union Operational Programme “Innovative Economy” Award Number: POIG.01.01.02-14-072/09. The funder provided support in the form of salaries for OZ-S, IG, MDu, MK, DM, KK, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section. 2. Foundation for Polish Science, Award Number: HOMING PLUS/2011-3/1. The funder provided support in the form of salaries for DD, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section. 3. Foundation for Polish Science, Award Number: Bridge/2013-8/8. The funder provided support in the form of salaries for IG and KK, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.
                Categories
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                Biology and Life Sciences
                Biochemistry
                Lipids
                Cholesterol
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