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oxLDL increases endothelial stiffness, force generation and network formation
14th IVBM Abstracts
A10.05 Nox1 mediates basic fibroblast growth factor-induced smooth 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 vascular smooth muscle cells (VSMC) migration. We determined whether bFGF-induced cell migration requires the formation of reactive oxygen species (ROS) and studied the underlying mechanisms. bFGF rapidly increased smooth muscle ROS formation via a mechanism sensitive to the specific NADPH oxidase inhibitors apocynin and gp91ds-tat as well as to inhibition of PI3-kinase and Rac. Pre-treatment of SMC with the NADPH oxidase inhibitor gp91ds-tat also completely prevented the bFGFinduced activation of p38 MAP kinase and JNK. bFGF-induced a marked migration of VSMC, which was blocked by PI3-kinase and Rac inhibitors. Moreover antioxidants as well as the specific inhibition of the NADPH oxidase prevented migration. In order to determine the NADPH oxidase homologues responsible for bFGF-induced migration, siRNA against Nox homologues were used. bFGF-induced migration was completely blocked by siRNA directed against Nox1 but remained unaffected by Nox4 siRNA. Also Nox1 siRNA, but not Nox4 or scr siRNA impaired bFGF-induced phosphorylation of tyrosine residues and JNK. These data demonstrate that bFGF activates the Nox1-containing NADPH oxidase via a pathway involving Rac and PI3-Kinase. Nox1-dependent ROS formation is critical for bFGF-induced signaling leading to VSMC migration.
e73
both properties playing important roles in the interaction of endothelial cells with the extracellular matrix, migration and morphogenesis. Our data show that exposing HAECs to 10– 50 νg/ml oxLDL for 1–6 h results in a 90% decrease in membrane deformability of HAECs as determined by micropipette aspiration. Furthermore, aortic endothelial cells freshly isolated from hypercholesterolemic pigs (PAECs) were significantly stiffer than cells isolated from healthy animals. Interestingly, oxLDL had no effect on membrane cholesterol of HAECs, but caused disappearance of a lipid raft marker, GM1, from the plasma membrane. Both, an increase in membrane stiffness and a disappearance of GM1 were also observed in cells that were cholesterol depleted by methyl-β-cyclodextrin (MβCD). Additionally, oxLDL treatment of HAECs embedded within collagen gels resulted in increased gel contraction, indicating an increase in force generation by the cells. This increase in force generation correlated with an increased ability of HAECs to elongate and form networks in a 3D environment. Increased force generation, elongation and network formation was also observed in cholesterol depleted cells. We suggest, therefore, that exposure to oxLDL results in disruption or redistribution of lipid rafts, which in turn induces stiffening of the endothelium, an increase in endothelial 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
doi:10.1016/j.vph.2006.08.205
A10.08 A10.06 oxLDL increases endothelial stiffness, force generation and network formation 1
2
1
Fitzroy J. Byfield , George H. Rothblat , Keith J. Gooch , Irena Levitan1,3
Effect of anti-oxidative enzyme gene-transfer on endothelial cell function of apolipoprotein-E deficient mice P-J. Guns1, T. Van Assche1, W. Verreth2, P. Fransen1, B. Mackness3, M. Mackness3, P. Holvoet2, H. Bult1 1
University of Antwerp, Belgium, Antwerp, Belgium Katholieke Universiteit Leuven, Belgium, Leuven, Belgium 3 University of Manchester, UK, Manchester, United Kingdom
1
2
Oxidative damage of Low Density Lipoproteins (LDL) is well known to be associated with an increased risk for coronary artery disease and plaque-formation. This study investigates the effect of oxidatively modified LDL (oxLDL) on the biomechanical properties of human aortic endothelial cells (HAECs). Specifically, we focus on endothelial deformability and force generation,
Objective: Apolipoprotein-E deficient mice (apoE-/-) develop spontaneously atherosclerotic plaques at preference locations, which then display endothelial dysfunction, defined as impaired endothelium-dependent relaxations. This endothelial dysfunction might be due to elevated oxidative stress in the atherosclerotic plaque. Previously we have shown that longterm over-expression of human apolipoprotein A1, the main protein of HDL, preserved endothelial function by retarding plaque-formation. In this study, we investigated whether short-
University of Pennsylvania, Philadelphia, United States of America 2 Children's Hospital of Philadelphia, Philadelphia, United States of America 3 University of Illinois, Chicago, United States of America
A10.05 Nox1 mediates basic fibroblast growth factor-induced smooth 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 vascular smooth muscle cells (VSMC) migration. We determined whether bFGF-induced cell migration requires the formation of reactive oxygen species (ROS) and studied the underlying mechanisms. bFGF rapidly increased smooth muscle ROS formation via a mechanism sensitive to the specific NADPH oxidase inhibitors apocynin and gp91ds-tat as well as to inhibition of PI3-kinase and Rac. Pre-treatment of SMC with the NADPH oxidase inhibitor gp91ds-tat also completely prevented the bFGFinduced activation of p38 MAP kinase and JNK. bFGF-induced a marked migration of VSMC, which was blocked by PI3-kinase and Rac inhibitors. Moreover antioxidants as well as the specific inhibition of the NADPH oxidase prevented migration. In order to determine the NADPH oxidase homologues responsible for bFGF-induced migration, siRNA against Nox homologues were used. bFGF-induced migration was completely blocked by siRNA directed against Nox1 but remained unaffected by Nox4 siRNA. Also Nox1 siRNA, but not Nox4 or scr siRNA impaired bFGF-induced phosphorylation of tyrosine residues and JNK. These data demonstrate that bFGF activates the Nox1-containing NADPH oxidase via a pathway involving Rac and PI3-Kinase. Nox1-dependent ROS formation is critical for bFGF-induced signaling leading to VSMC migration.
e73
both properties playing important roles in the interaction of endothelial cells with the extracellular matrix, migration and morphogenesis. Our data show that exposing HAECs to 10– 50 νg/ml oxLDL for 1–6 h results in a 90% decrease in membrane deformability of HAECs as determined by micropipette aspiration. Furthermore, aortic endothelial cells freshly isolated from hypercholesterolemic pigs (PAECs) were significantly stiffer than cells isolated from healthy animals. Interestingly, oxLDL had no effect on membrane cholesterol of HAECs, but caused disappearance of a lipid raft marker, GM1, from the plasma membrane. Both, an increase in membrane stiffness and a disappearance of GM1 were also observed in cells that were cholesterol depleted by methyl-β-cyclodextrin (MβCD). Additionally, oxLDL treatment of HAECs embedded within collagen gels resulted in increased gel contraction, indicating an increase in force generation by the cells. This increase in force generation correlated with an increased ability of HAECs to elongate and form networks in a 3D environment. Increased force generation, elongation and network formation was also observed in cholesterol depleted cells. We suggest, therefore, that exposure to oxLDL results in disruption or redistribution of lipid rafts, which in turn induces stiffening of the endothelium, an increase in endothelial 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
doi:10.1016/j.vph.2006.08.205
A10.08 A10.06 oxLDL increases endothelial stiffness, force generation and network formation 1
2
1
Fitzroy J. Byfield , George H. Rothblat , Keith J. Gooch , Irena Levitan1,3
Effect of anti-oxidative enzyme gene-transfer on endothelial cell function of apolipoprotein-E deficient mice P-J. Guns1, T. Van Assche1, W. Verreth2, P. Fransen1, B. Mackness3, M. Mackness3, P. Holvoet2, H. Bult1 1
University of Antwerp, Belgium, Antwerp, Belgium Katholieke Universiteit Leuven, Belgium, Leuven, Belgium 3 University of Manchester, UK, Manchester, United Kingdom
1
2
Oxidative damage of Low Density Lipoproteins (LDL) is well known to be associated with an increased risk for coronary artery disease and plaque-formation. This study investigates the effect of oxidatively modified LDL (oxLDL) on the biomechanical properties of human aortic endothelial cells (HAECs). Specifically, we focus on endothelial deformability and force generation,
Objective: Apolipoprotein-E deficient mice (apoE-/-) develop spontaneously atherosclerotic plaques at preference locations, which then display endothelial dysfunction, defined as impaired endothelium-dependent relaxations. This endothelial dysfunction might be due to elevated oxidative stress in the atherosclerotic plaque. Previously we have shown that longterm over-expression of human apolipoprotein A1, the main protein of HDL, preserved endothelial function by retarding plaque-formation. In this study, we investigated whether short-
University of Pennsylvania, Philadelphia, United States of America 2 Children's Hospital of Philadelphia, Philadelphia, United States of America 3 University of Illinois, Chicago, United States of America