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Cyclophilin A differentially activates monocytes and endothelial cells Role of purity, activity, and endotoxin contamination in commercial preparations


Payeli, S K; Schiene-Fischer, C; Steffel, J; Camic, G G; Rozenberg, I; Lüscher, T F; Tanner, F C (2008). Cyclophilin A differentially activates monocytes and endothelial cells Role of purity, activity, and endotoxin contamination in commercial preparations. Atherosclerosis, 197(2):564-571.

Abstract

BACKGROUND: Cyclophilin A (CyPA) is a cytoplasmic protein secreted under inflammatory conditions. Extracellular CyPA is detected in atherosclerotic plaques and has been observed to activate endothelial cells as well as monocytes. METHODS AND RESULTS: Commercially available recombinant CyPA-induced expression of tissue factor (TF) and vascular cell adhesion molecule-1 (VCAM-1) in human aortic endothelial cells (HAEC). However, CyPA from commercial sources contained lipopolysaccharide at concentrations up to 18.9 ng/ml; moreover, it exhibited low purity as determined by protein spectrum analysis and low activity as assessed by peptidyl prolyl cis-trans isomerase (PPIase) assay. An in-house preparation of pure, active, and uncontaminated CyPA failed to induce endothelial TF or VCAM-1 expression; moreover, it was not chemotactic for HAEC. In contrast, such CyPA exhibited potent chemotactic activity on monocytic THP-1 cells, with a maximal effect on migration occurring at a concentration of 5.5 x 10(-9)mol/l. Pretreatment of CyPA with cyclosporine A prevented its effect on THP-1 cell migration; similarly, PPIase-deficient mutant CyPA protein did not induce migration of these cells. In-house prepared CyPA induced the release of Il-6, but not TNF-alpha, from THP-1 cells. CONCLUSIONS: Commercially available CyPA exhibits low purity and activity and may be contaminated by endotoxin. Pure, active, and uncontaminated CyPA does not induce endothelial TF or VCAM-1 expression; instead, it acts as a potent monocyte chemoattractant and induces monocyte Il-6 release, implying a role for extracellular CyPA in the pathogenesis of atherosclerosis via activation of monocytes rather than endothelial cells.

BACKGROUND: Cyclophilin A (CyPA) is a cytoplasmic protein secreted under inflammatory conditions. Extracellular CyPA is detected in atherosclerotic plaques and has been observed to activate endothelial cells as well as monocytes. METHODS AND RESULTS: Commercially available recombinant CyPA-induced expression of tissue factor (TF) and vascular cell adhesion molecule-1 (VCAM-1) in human aortic endothelial cells (HAEC). However, CyPA from commercial sources contained lipopolysaccharide at concentrations up to 18.9 ng/ml; moreover, it exhibited low purity as determined by protein spectrum analysis and low activity as assessed by peptidyl prolyl cis-trans isomerase (PPIase) assay. An in-house preparation of pure, active, and uncontaminated CyPA failed to induce endothelial TF or VCAM-1 expression; moreover, it was not chemotactic for HAEC. In contrast, such CyPA exhibited potent chemotactic activity on monocytic THP-1 cells, with a maximal effect on migration occurring at a concentration of 5.5 x 10(-9)mol/l. Pretreatment of CyPA with cyclosporine A prevented its effect on THP-1 cell migration; similarly, PPIase-deficient mutant CyPA protein did not induce migration of these cells. In-house prepared CyPA induced the release of Il-6, but not TNF-alpha, from THP-1 cells. CONCLUSIONS: Commercially available CyPA exhibits low purity and activity and may be contaminated by endotoxin. Pure, active, and uncontaminated CyPA does not induce endothelial TF or VCAM-1 expression; instead, it acts as a potent monocyte chemoattractant and induces monocyte Il-6 release, implying a role for extracellular CyPA in the pathogenesis of atherosclerosis via activation of monocytes rather than endothelial cells.

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Item Type:Journal Article, refereed, further contribution
Communities & Collections:04 Faculty of Medicine > Institute of Physiology
07 Faculty of Science > Institute of Physiology
Dewey Decimal Classification:570 Life sciences; biology
Language:English
Date:2008
Deposited On:19 Jan 2009 17:17
Last Modified:05 Apr 2016 12:51
Publisher:Elsevier
ISSN:0021-9150
Publisher DOI:10.1016/j.atherosclerosis.2007.08.025
PubMed ID:17919644
Permanent URL: http://doi.org/10.5167/uzh-10698

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