title: A combined impedance and AlphaLISA-based approach to identify anti-inflammatory and barrier-protective compounds in human endothelium.
abstract: Chronic inflammation is at least partially mediated by the chemokine-mediated attraction and by the adhesion molecule-directed binding of leukocytes to the activated endothelium. Therefore, it is therapeutically important to identify anti-inflammatory compounds able to control the interaction between leukocytes and the endothelial compartments of the micro- and macrocirculation. When testing novel drug candidates, it is, however, of the utmost importance to detect side effects, such as potential cytotoxic and barrier-disruptive activities. Indeed, minor changes in the endothelial monolayer integrity may increase the permeability of small blood vessels and capillaries, which, in extreme cases, can lead to edema development. Here, we describe the development of a high-throughput screening (HTS) platform, based on AlphaLISA technology, able to identify anti-inflammatory nontoxic natural or synthetic compounds capable of reducing tumor necrosis factor (TNF)-induced chemokine (interleukin [IL]-8) and adhesion molecule (ICAM-1) expression in human lung microvascular endothelial cells. Quantification of cell membrane-expressed ICAM-1 and of cell culture supernatant-associated levels of IL-8 was analyzed in HTS. In parallel, we monitored monolayer integrity and endothelial cell viability using the electrical cell substrate impedance sensing method. This platform allowed us to identify natural secondary metabolites from cyanobacteria, capable of reducing ICAM-1 and IL-8 levels in TNF-activated human microvascular endothelial cells in the absence of endothelial monolayer barrier disruption.
authors.affiliation: Department of Medical and Pharmaceutical Biotechnology, University of Applied Sciences, Krems, Austria. maren.pflueger@fh-krems.ac.at
authors.forename: Maren
authors.initials: M
authors.lastname: Pflüger
authors.forename: Aleksandra
authors.initials: A
authors.lastname: Kapuscik
authors.forename: Rudolf
authors.initials: R
authors.lastname: Lucas
authors.forename: Anita
authors.initials: A
authors.lastname: Koppensteiner
authors.forename: Michael
authors.initials: M
authors.lastname: Katzlinger
authors.forename: Jouni
authors.initials: J
authors.lastname: Jokela
authors.forename: Andreas
authors.initials: A
authors.lastname: Eger
authors.forename: Nico
authors.initials: N
authors.lastname: Jacobi
authors.forename: Christoph
authors.initials: C
authors.lastname: Wiesner
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Hofmann
authors.forename: Kamil
authors.initials: K
authors.lastname: Onder
authors.forename: Jiri
authors.initials: J
authors.lastname: Kopecky
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Schütt
authors.forename: Harald
authors.initials: H
authors.lastname: Hundsberger
mesh_terms: D000893:Anti-Inflammatory Agents; D002199:Capillary Permeability / Q000187:drug effects; D015815:Cell Adhesion Molecules / Q000235:genetics / Q000378:metabolism; D000458:Cyanobacteria / Q000737:chemistry; D016207:Cytokines / Q000235:genetics / Q000378:metabolism; D047630:Depsipeptides / Q000302:isolation & purification / Q000494:pharmacology; D017097:Electric Impedance; D042783:Endothelial Cells / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology; D004730:Endothelium, Vascular / Q000187:drug effects; D015870:Gene Expression / Q000187:drug effects; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D018799:Intercellular Adhesion Molecule-1 / Q000235:genetics / Q000378:metabolism; D016209:Interleukin-8 / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008168:Lung / Q000098:blood supply; D055806:Microvessels / Q000166:cytology; D010456:Peptides, Cyclic / Q000302:isolation & purification / Q000494:pharmacology; D011725:Pyridines / Q000494:pharmacology; D015203:Reproducibility of Results; D015533:Transcriptional Activation / Q000187:drug effects; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology / Q000502:physiology
chemical_list: D000893:Anti-Inflammatory Agents; D015815:Cell Adhesion Molecules; D016207:Cytokines; D047630:Depsipeptides; D007093:Imidazoles; D016209:Interleukin-8; D010456:Peptides, Cyclic; D011725:Pyridines; D014409:Tumor Necrosis Factor-alpha; D018799:Intercellular Adhesion Molecule-1; C093642:SB 203580