Hemodynamic Regulation of Inflammation at the Endothelial–Neutrophil Interface

ArticleinAnnals of Biomedical Engineering 36(4):586-95 · May 2008with11 Reads
DOI: 10.1007/s10439-008-9465-4 · Source: PubMed
Arterial shear stress can regulate endothelial phenotype. The potential for anti-inflammatory effects of shear stress on TNFalpha-activated endothelium was tested in assays of cytokine expression and neutrophil adhesion. In cultured human aortic endothelial cells (HAEC), arterial shear stress of 10 dyne/cm(2) blocked by >80% the induction by 5 ng/mL TNFalpha of interleukin-8 (IL-8) and IL-6 secretion (50 and 90% reduction, respectively, in the presence of nitric oxide synthase antagonism with 200 microM nitro-L-arginine methylester, L-NAME). Exposure of TNFalpha-stimulated HAEC to arterial shear stress for 5 h also reduced by 60% (p < 0.001) the conversion of neutrophil rolling to firm arrest in a venous flow assay conducted at 1 dyne/cm(2). Also, neutrophil rolling lengths at 1 dyne/cm(2) were longer when TNFalpha-stimulated HAEC were presheared for 5 h at arterial stresses. In experiments with a synthetic promoter that provides luciferase induction to detect cis interactions of glucocorticoid receptor (GR) and NFkappaB, shear stress caused a marked 40-fold induction of luciferase in TNFalpha-treated cells, suggesting a role for GR pathways in the anti-inflammatory actions of fluid shear stress. Hemodynamic force exerts anti-inflammatory effects on cytokine-activated endothelium by attenuation of cytokine expression and neutrophil firm arrest.
    • "We show for the first time that the co-stimulation of naı¨venaı¨ve ECs (not previously exposed to shear in vitro) with flow-induced shear and IL-1b induces the expression of significantly higher levels of functional E-selectin molecules up to 24 hr when compared with monolayers stimulated under static condition only (Figure 1). This is interesting since E-selectin expression by ECs is typically not inducible by laminar shear alone [35,47,48] as was shown inFigure 1 though one report showed a 4 fold increase in E-selectin mRNA in human aortic ECs in response to 4.5 dyn cm 22 (but not at .10 dyn cm 22 ) of laminar shear [49]. Similarly, peak E-selectin expression is well-documented to occur between 4–6 hr in staticcytokine stimulated cells compared to the 8–12 hr peak observed with naı¨venaı¨ve cells under shear-cytokine activation (Figure 2A). "
    [Show abstract] [Hide abstract] ABSTRACT: Endothelial cells (ECs) are continuously exposed to hemodynamic forces imparted by blood flow. While it is known that endothelial behavior can be influenced by cytokine activation or fluid shear, the combined effects of these two independent agonists have yet to be fully elucidated. We investigated EC response to long-term inflammatory cues under physiologically relevant shear conditions via E-selectin expression where monolayers of human umbilical vein ECs were simultaneously exposed to laminar fluid shear and interleukin-1ß (shear-cytokine activation) in a parallel plate flow chamber. Naïve ECs exposed to shear-cytokine activation display significantly higher E-selectin expression for up to 24 hr relative to ECs activated in static (static-cytokine). Peak E-selectin expression occurred after 8-12 hr of continuous shear-cytokine activation contrary to the commonly observed 4-6 hr peak expression in ECs exposed to static-cytokine activation. Cells with some history of high shear conditioning exhibited either high or muted E-selectin expression depending on the durations of the shear pre-conditioning and the ensuing shear-cytokine activation. Overall, the presented data suggest that a high laminar shear enhances acute EC response to interleukin-1ß in naïve or shear-conditioned ECs as may be found in the pathological setting of ischemia/reperfusion injury while conferring rapid E-selectin downregulation to protect against chronic inflammation.
    Full-text · Article · Feb 2012
    • "The expression of adhesion molecules, such as VCAM-1, in response to chemokines and cytokines is essential in the acute inflammatory response and represents a clear sign of an activated endothelial phenotype [35,373839. Unidimensional and bidimensional western blots analy- sis [40,41]Figure 2A). "
    [Show abstract] [Hide abstract] ABSTRACT: Several common aspects of endothelial phenotype, such as the expression of cell adhesion molecules, are shared between metastasis and inflammation. Here, we analyzed VCAM-1 variants as biological markers of these two types of endothelial cell activation. With the combination of 2-DE and western blot techniques and the aid of tunicamycin, we analyzed N-glycosylation variants of VCAM-1 in primary human endothelial cells stimulated with either TNF or tumoral soluble factors (TSF's) derived from the human breast cancer cell line ZR75.30. Treatments induced a pro-adhesive endothelial phenotype. 2D western blots analysis of cells subjected to both treatments revealed the expression of the two known VCAM-1 isoforms and of previously unknown isoforms. In particular TSFZR75.30 induced an isoform with a relative molecular mass (Mr) and isoelectric point (pI) of 75-77 kDa and 5.0, respectively. The unknown isoforms of VCAM-1 that were found to be overexpressed after treatment with TSF's compared with TNF, could serve as biomarkers to discriminate between inflammation and metastasis. 2D western blots revealed three new VCAM-1 isoforms expressed in primary human endothelial cells in response to TSF stimulation. Each of these isoforms varies in Mr and pI and could be the result of differential glycosylation states.
    Full-text · Article · Nov 2009
    • "EndothelzellenCotran 1987; Hecker 1999; Ji, Jing et al. 2008). Aufgrund dieser Tatsache ist es leicht zu verstehen, dass gestörte "
    [Show abstract] [Hide abstract] ABSTRACT: Das Gefäßendothel nimmt als homogene Zellschicht neben der Regulation von Gefäßtonus, Blutfließeigenschaften sowie der Koagulation und Thrombusbildung eine bedeutende Rolle in der Steuerung der Barrierefunktion ein. Intermedin, ein 2004 entdecktes neues Mitglied der Calcitonin-Peptid-Familie spielt eine wichtige Rolle in der Regulation der endothelialen Barrierefunktion. Ziel dieser Arbeit war die Identifizierung des molekularen Mechanismus, über den Intermedin die Schrankenfunktion beeinflusst. Im Mittelpunkt der Untersuchungen standen dabei die Fragen nach der Signaltransduktion und der Wirkung, welche Intermedin auf das endotheliale Zytoskelett hat. Zur Beantwortung dieser Fragen wurden an kultivierten einschichtigen Ratten-Endothelzellen die Wirkung von Intermedin auf die endotheliale Permeabilität, CGRP-Rezeptoren, RhoA, ROCK, das Aktin-Zytoskelett und die kontraktile Aktivierung untersucht. Stimulation von Endothelzellen mikrovaskulären Ursprungs mit Intermedin führte bereits bei einer Konzentration von 0,1 nM zu einer signifikanten Steigerung der Makromolekülpermeabilität, begleitet von einer Abnahme der RhoA-Aktivität sowie der kontraktilen Aktivität. Durch den gezielten Einsatz des panspezifischen IMD-Rezeptorantagonisten alfa-CGRP8-37 und des PKA-Hemmstoffs H89 konnte gezeigt werden, dass Intermedin seine Wirkung auf die intrazelluläre Signaltransduktion über spezifische CRLR/RAMP-Rezeptor-Komplexe vermittelt und dabei Endothelzellen über den cAMP/PKA-Weg aktiviert. Ebenfalls fand sich nach Gabe des Rho-Inhibitors C3T oder des ROCK-Inhibitors Y27632 eine dem Intermedin äquipotente Wirkung auf die endotheliale Permeabilität. Des Weiteren konnte gezeigt werden, dass Intermedin über eine Dephosphorylierung des Aktin-bindenden-Proteins Kofilin die Deassemblierung von F-Aktin und somit eine Destabilisierung des Aktinzytoskeletts bewirkt. Damit präsentiert diese Arbeit Intermedin als einen neuen potenten Regulator der endothelialen Barrierefunktion. The vascular endothelium is a homogenous cell layer and plays a considerable role in the control of endothelial barrier function, regulation of the vascular tone, characteristics of blood flow, as well as coagulation and formation of blood clots. Intermedin was discovered in 2004 as a new member of the calcitonin / CGRP family, and it has a significant influence on the regulation of the endothelial barrier function. The aim of this study was to identify the molecular mechanisms through which intermedin affects the vascular endothelium. The focus of the investigations was laid on the signalling pathways which are activated by intermedin and their effect on the endothelial cytoskeleton. In order to answer this questions, rodent endothelial cells were exposed to intermedin, in vitro, and the changes in the endothelial permeability, the CGRP-receptor, RhoA, ROCK, the actin cytoskeleton and the contractile activation were analysed. Exposure to intermedin led to a rapid increase in the endothelial macromolecule permeability already at a concentration of 0.1 nM. Intermedin also induced a reduction in RhoA and contractile activity. Usage of the pan-specific intermedin receptor antagonist alfa-CGRP8-37 and the protein kinase inhibitor H89 revealed that intermedin mediates its effect on the intracellular signal transduction via specific CRLR/RAMP receptor complexes which then activate endothelial cells via the cAMP/PKA pathway. Furthermore, administration of the Rho-inhibitor C3-transferase and the ROCK-inhibitor Y27632 demonstrated an equally potent effect on endothelial permeability as intermedin. In addition, intermedin caused a depletion of actin fibres via dephosphorylation of the actin-binding-protein Cofilin causing the destabilization of the actin cytoskeleton. Thus, the present study reveals intermedin as a new potent regulator of the endothelial barrier function.
    Article · · BMC Chemical Biology
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