Role of reactive oxygen species in inhibition of endothelial cell migration by oxidized low-density lipoprotein  John A. van Aalst, MD, Dong-Mei Zhang, PhD, Keiko Miyazaki, MD, Scott M. Colles, PhD, Paul L. Fox, PhD, Linda M. Graham, MD  Journal of Vascular Surgery  Volume 40, Issue 6, Pages 1208-1215 (December 2004) DOI: 10.1016/j.jvs.2004.09.020 Copyright © 2004 The Society for Vascular Surgery Terms and Conditions

Fig 1 Agonist-induced reactive oxygen species production paralleled inhibition of endothelial cell migration. A, Reactive oxygen species generation was determined with fluorescence microscopy (see Methods). Representative results of 1 of 3 separate experiments are shown. B, In parallel studies, endothelial cell migration was assessed with the razor scrape assay. Migration was stopped at 24 hours by fixing cells with Wright-Giemsa stain. Arrow indicates starting line for migration. Substances tested included culture medium, oxidized low-density lipoprotein (oxLDL; 400 μg cholesterol/mL), lysophosphatidyl phosphatidylcholine (lysoPC, 10 μmol/L), 6-anilinoquinoline-5,8,quinone (LY83583, 2.5 μmol/L), or 2,3-dimethoxy-1,4-naphthoquinone (DMNQ, 7.5 μmol/L). LDL, Low-density lipoprotein. Journal of Vascular Surgery 2004 40, 1208-1215DOI: (10.1016/j.jvs.2004.09.020) Copyright © 2004 The Society for Vascular Surgery Terms and Conditions

Fig 2 Superoxide dismutase (SOD) preserved migration of endothelial cells incubated with oxidized low-density lipoprotein (oxLDL) or lysophosphtidyl phosphatidylcholine (lysoPC). A, Reactive oxygen species generation was determined as described (see Methods). SOD was added before the agonists indicated. Representative results of 3 separate experiments are shown. B, Endothelial cells were grown to confluence, then incubated with SOD (0, 50, 150 U/mL) for 1 hour. Migration assay was initiated, then SOD and oxLDL (400 μg cholesterol/mL) were added. Endothelial cells were allowed to migrate for 24 hours, then migration was terminated by fixing cells with Wright-Giemsa stain. C, Number of endothelial cells (EC) crossing starting line was quantitated with image analysis. D, Ability of SOD to decrease lysoPC-induced reactive oxygen species production was assessed as described. E, Similarly, ability of SOD (50 U/mL) to preserve endothelial cell migration in lysoPC (5 or 10 μmol/L) was studied. F, Migration at 24 hours was quantitated with image analysis. *P ≤ .05 vs medium, **P < .01 vs medium. Statistical difference between endothelial cell migration with or without SOD is shown in boxed insets. Journal of Vascular Surgery 2004 40, 1208-1215DOI: (10.1016/j.jvs.2004.09.020) Copyright © 2004 The Society for Vascular Surgery Terms and Conditions

Fig 3 Scavengers of −OH and hydrogen peroxide failed to preserve migration in presence of oxidized low-density lipoprotein (oxLDL). Confluent monolayers of endothelial cells were incubated for 1 hour with mannitol (10 mmol/L), glutathione (1 mmol/L), or catalase (300 U/mL); then razor scrape was performed. Endothelial cells were washed and oxLDL (400 μg cholesterol/mL), and mannitol, glutathione, or catalase was again added. Migration was terminated after 24 hours with Wright-Giemsa stain. LDL, Low-density lipoprotein. Journal of Vascular Surgery 2004 40, 1208-1215DOI: (10.1016/j.jvs.2004.09.020) Copyright © 2004 The Society for Vascular Surgery Terms and Conditions

Fig 4 Cyclooxygenase and xanthine oxidase inhibitors did not preserve endothelial cell migration in oxidized low-density lipoprotein (oxLDL). A, Indomethacin, a cyclooxygenase inhibitor, was incubated with confluent monolayers of endothelial cells (EC) for 1 hour. Migration was initiated in the razor scrape assay, and oxLDL (400 μg cholesterol/mL) and the inhibitor were added. Endothelial cells were allowed to migrate for 24 hours. Migration was terminated with addition of Wright-Giemsa stain, and number of cells crossing starting line was quantitated with image analysis. B, Effect of allopurinol, a xanthine oxidase inhibitor on endothelial cell migration in oxLDL, was assessed. *P < .05 vs medium, **P ≤ .01 vs medium. Journal of Vascular Surgery 2004 40, 1208-1215DOI: (10.1016/j.jvs.2004.09.020) Copyright © 2004 The Society for Vascular Surgery Terms and Conditions

Fig 5 Reduced nicotinamide adenine dinucleotide (phosphate) (NAD[P]H) oxidase inhibitors inhibited endothelial cell production of reactive oxygen species and preserved endothelial cell migration in oxidized low-density lipoprotein (oxLDL). A, Endothelial cells were made quiescent overnight, then diphenylene iodonium (DPI, 0.5 μmol/L), quinacrine (Q, 1 μmol/L), or hydralazine (Hyd, 25 μmol/L) was added. After 30 minutes dichlorofluorescein was added for 30 minutes, then endothelial cells were incubated with oxLDL for 3 hours. Reactive oxygen species generation was assessed with fluorescence microscopy. Representative results of 1 of 3 separate experiments are shown. B, In parallel studies, migration was assessed after endothelial cells (EC) were preincubated with NAD(P)H oxidase inhibitors for 1 hour. After razor scrape, NAD(P)H oxidase inhibitor was added along with oxLDL (400 μg cholesterol/mL). Migration was stopped at 24 hours with addition of Wright-Giemsa stain, and quantitated with image analysis. *P < .05 vs medium, **P ≤ .01 vs medium. Journal of Vascular Surgery 2004 40, 1208-1215DOI: (10.1016/j.jvs.2004.09.020) Copyright © 2004 The Society for Vascular Surgery Terms and Conditions