e7314th IVBM Abstracts

A10.05

Nox1 mediates basic fibroblast growth factor-inducedsmooth muscle cells migration

Katrin Schröder, Alexandra Keller, Rudi Busse,Ralf P. Brandes

J.W. Goethe-Universität, Frankfurt am Main, Germany

Basic fibroblast growth factor (bFGF) induces vascularsmoothmuscle cells (VSMC)migration.We determinedwhetherbFGF-induced cell migration requires the formation of reactiveoxygen species (ROS) and studied the underlying mechanisms.bFGF rapidly increased smooth muscle ROS formation via amechanism sensitive to the specific NADPH oxidase inhibitorsapocynin and gp91ds-tat as well as to inhibition of PI3-kinaseand Rac. Pre-treatment of SMC with the NADPH oxidaseinhibitor gp91ds-tat also completely prevented the bFGF-induced activation of p38 MAP kinase and JNK. bFGF-induceda marked migration of VSMC, which was blocked by PI3-kinaseand Rac inhibitors. Moreover antioxidants as well as the specificinhibition of the NADPH oxidase prevented migration. In orderto determine the NADPH oxidase homologues responsible forbFGF-induced migration, siRNA against Nox homologues wereused. bFGF-induced migration was completely blocked bysiRNA directed against Nox1 but remained unaffected by Nox4siRNA. Also Nox1 siRNA, but not Nox4 or scr siRNA impairedbFGF-induced phosphorylation of tyrosine residues and JNK.These data demonstrate that bFGF activates the Nox1-containingNADPH oxidase via a pathway involving Rac and PI3-Kinase.Nox1-dependent ROS formation is critical for bFGF-inducedsignaling leading to VSMC migration.

doi:10.1016/j.vph.2006.08.205

A10.06

oxLDL increases endothelial stiffness, force generation andnetwork formation

Fitzroy J. Byfield1, George H. Rothblat2, Keith J. Gooch1,Irena Levitan1,3

1University of Pennsylvania, Philadelphia,United States of America2Children's Hospital of Philadelphia, Philadelphia,United States of America3University of Illinois, Chicago, United States of America

Oxidative damage of LowDensity Lipoproteins (LDL) is wellknown to be associated with an increased risk for coronary arterydisease and plaque-formation. This study investigates the effectof oxidatively modified LDL (oxLDL) on the biomechanicalproperties of human aortic endothelial cells (HAECs). Specifi-cally, we focus on endothelial deformability and force generation,

both properties playing important roles in the interaction ofendothelial cells with the extracellular matrix, migration andmorphogenesis. Our data show that exposing HAECs to 10–50 νg/ml oxLDL for 1–6 h results in a 90% decrease inmembrane deformability of HAECs as determined by micropi-pette aspiration. Furthermore, aortic endothelial cells freshlyisolated from hypercholesterolemic pigs (PAECs) were signifi-cantly stiffer than cells isolated from healthy animals. Interest-ingly, oxLDL had no effect on membrane cholesterol of HAECs,but caused disappearance of a lipid raft marker, GM1, from theplasma membrane. Both, an increase in membrane stiffness and adisappearance of GM1 were also observed in cells that werecholesterol depleted by methyl-β-cyclodextrin (MβCD). Addi-tionally, oxLDL treatment of HAECs embedded within collagengels resulted in increased gel contraction, indicating an increasein force generation by the cells. This increase in force generationcorrelated with an increased ability of HAECs to elongate andform networks in a 3D environment. Increased force generation,elongation and network formation was also observed incholesterol depleted cells. We suggest, therefore, that exposureto oxLDL results in disruption or redistribution of lipid rafts,which in turn induces stiffening of the endothelium, an increase inendothelial force generation and potential for network formation.

doi:10.1016/j.vph.2006.08.206

A10.07

Withdrawn

doi:10.1016/j.vph.2006.08.207

A10.08

Effect of anti-oxidative enzyme gene-transfer onendothelial cell function of apolipoprotein-Edeficient mice

P-J. Guns1, T. Van Assche1, W. Verreth2, P. Fransen1,B. Mackness3, M. Mackness3, P. Holvoet2, H. Bult1

1University of Antwerp, Belgium, Antwerp, Belgium2Katholieke Universiteit Leuven, Belgium, Leuven, Belgium3University of Manchester, UK, Manchester, United Kingdom

Objective: Apolipoprotein-E deficient mice (apoE-/-) developspontaneously atherosclerotic plaques at preference locations,which then display endothelial dysfunction, defined as impairedendothelium-dependent relaxations. This endothelial dysfunc-tion might be due to elevated oxidative stress in theatherosclerotic plaque. Previously we have shown that long-term over-expression of human apolipoprotein A1, the mainprotein of HDL, preserved endothelial function by retardingplaque-formation. In this study, we investigated whether short-