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Prasad V. Gade, MD, José A. Andrades, PhD, Marcel E

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1 Nitric oxide mediates hyperglycemia-induced defective migration in cultured endothelial cells 
Prasad V. Gade, MD, José A. Andrades, PhD, Marcel E. Nimni, PhD, José Becerra, PhD, James Longoria, MD, Nadereh Asemanfar, BA, Nino Sorgente, PhD  Journal of Vascular Surgery  Volume 26, Issue 2, Pages (August 1997) DOI: /S (97) Copyright © 1997 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

2 Fig. 1 Linear relationship between cell number and total cellular protein in the cultures. Journal of Vascular Surgery  , DOI: ( /S (97) ) Copyright © 1997 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

3 Fig. 2 Effect of 5-FU on endothelial cell proliferation. It is evident that 5-FU inhibits proliferation at all concentrations used. Journal of Vascular Surgery  , DOI: ( /S (97) ) Copyright © 1997 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

4 Fig. 3 Micrographs of the modulation of cell migration by glucose and NO. Direction of cell migration is toward top of page. A, In the presence of 5.5 mmol/L glucose, endothelial cells migrate as a sheet with few single, rounded cells detached from leading edge. B, Increasing glucose concentration to 27.7 mmol/L results in marked inhibition of migration. Cell sheet is not evident; however, some rounded cells have migrated onto denuded area of dish. C, Treating cultures in the presence of 27.7 mmol/L glucose with NMMA (0.15 mmol/L), an inhibitor of NO synthase, abolishes inhibitory effect of elevated glucose. D, NMMA also abolishes inhibitory effects of 16.5 mmol/L glucose (not shown); on the other hand, addition of S-nitrosylated penicillamine, a NO donor, in the presence of 5.5 mmol/L glucose results in inhibition of migration. Original magnification, ×100. Journal of Vascular Surgery  , DOI: ( /S (97) ) Copyright © 1997 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

5 Fig. 3 Micrographs of the modulation of cell migration by glucose and NO. Direction of cell migration is toward top of page. A, In the presence of 5.5 mmol/L glucose, endothelial cells migrate as a sheet with few single, rounded cells detached from leading edge. B, Increasing glucose concentration to 27.7 mmol/L results in marked inhibition of migration. Cell sheet is not evident; however, some rounded cells have migrated onto denuded area of dish. C, Treating cultures in the presence of 27.7 mmol/L glucose with NMMA (0.15 mmol/L), an inhibitor of NO synthase, abolishes inhibitory effect of elevated glucose. D, NMMA also abolishes inhibitory effects of 16.5 mmol/L glucose (not shown); on the other hand, addition of S-nitrosylated penicillamine, a NO donor, in the presence of 5.5 mmol/L glucose results in inhibition of migration. Original magnification, ×100. Journal of Vascular Surgery  , DOI: ( /S (97) ) Copyright © 1997 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

6 Fig. 3 Micrographs of the modulation of cell migration by glucose and NO. Direction of cell migration is toward top of page. A, In the presence of 5.5 mmol/L glucose, endothelial cells migrate as a sheet with few single, rounded cells detached from leading edge. B, Increasing glucose concentration to 27.7 mmol/L results in marked inhibition of migration. Cell sheet is not evident; however, some rounded cells have migrated onto denuded area of dish. C, Treating cultures in the presence of 27.7 mmol/L glucose with NMMA (0.15 mmol/L), an inhibitor of NO synthase, abolishes inhibitory effect of elevated glucose. D, NMMA also abolishes inhibitory effects of 16.5 mmol/L glucose (not shown); on the other hand, addition of S-nitrosylated penicillamine, a NO donor, in the presence of 5.5 mmol/L glucose results in inhibition of migration. Original magnification, ×100. Journal of Vascular Surgery  , DOI: ( /S (97) ) Copyright © 1997 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

7 Fig. 3 Micrographs of the modulation of cell migration by glucose and NO. Direction of cell migration is toward top of page. A, In the presence of 5.5 mmol/L glucose, endothelial cells migrate as a sheet with few single, rounded cells detached from leading edge. B, Increasing glucose concentration to 27.7 mmol/L results in marked inhibition of migration. Cell sheet is not evident; however, some rounded cells have migrated onto denuded area of dish. C, Treating cultures in the presence of 27.7 mmol/L glucose with NMMA (0.15 mmol/L), an inhibitor of NO synthase, abolishes inhibitory effect of elevated glucose. D, NMMA also abolishes inhibitory effects of 16.5 mmol/L glucose (not shown); on the other hand, addition of S-nitrosylated penicillamine, a NO donor, in the presence of 5.5 mmol/L glucose results in inhibition of migration. Original magnification, ×100. Journal of Vascular Surgery  , DOI: ( /S (97) ) Copyright © 1997 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

8 Fig. 4 Fluorescence micrographs of cells stained for F-actin with fluorescein phalloidin 48 hours after wounding. In the presence of 5.5 mmol/L glucose (A) elongated endothelial cells exhibit prominent cytoplasmic stress fibers; treatment with NMMA did not alter this appearance (B). Cells at wound edge of cultures treated with 27.7 mmol/L of glucose had no stress fibers, but a condensed band of fluorescence was evident in perinuclear region (C), but when they were treated with NMMA, some stress fibers became apparent in cytoplasm and a prominent dense band of fluorescence (arrowheads) was evident at cell periphery (D). Original magnification, ×400. Journal of Vascular Surgery  , DOI: ( /S (97) ) Copyright © 1997 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

9 Fig. 4 Fluorescence micrographs of cells stained for F-actin with fluorescein phalloidin 48 hours after wounding. In the presence of 5.5 mmol/L glucose (A) elongated endothelial cells exhibit prominent cytoplasmic stress fibers; treatment with NMMA did not alter this appearance (B). Cells at wound edge of cultures treated with 27.7 mmol/L of glucose had no stress fibers, but a condensed band of fluorescence was evident in perinuclear region (C), but when they were treated with NMMA, some stress fibers became apparent in cytoplasm and a prominent dense band of fluorescence (arrowheads) was evident at cell periphery (D). Original magnification, ×400. Journal of Vascular Surgery  , DOI: ( /S (97) ) Copyright © 1997 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

10 Fig. 4 Fluorescence micrographs of cells stained for F-actin with fluorescein phalloidin 48 hours after wounding. In the presence of 5.5 mmol/L glucose (A) elongated endothelial cells exhibit prominent cytoplasmic stress fibers; treatment with NMMA did not alter this appearance (B). Cells at wound edge of cultures treated with 27.7 mmol/L of glucose had no stress fibers, but a condensed band of fluorescence was evident in perinuclear region (C), but when they were treated with NMMA, some stress fibers became apparent in cytoplasm and a prominent dense band of fluorescence (arrowheads) was evident at cell periphery (D). Original magnification, ×400. Journal of Vascular Surgery  , DOI: ( /S (97) ) Copyright © 1997 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

11 Fig. 4 Fluorescence micrographs of cells stained for F-actin with fluorescein phalloidin 48 hours after wounding. In the presence of 5.5 mmol/L glucose (A) elongated endothelial cells exhibit prominent cytoplasmic stress fibers; treatment with NMMA did not alter this appearance (B). Cells at wound edge of cultures treated with 27.7 mmol/L of glucose had no stress fibers, but a condensed band of fluorescence was evident in perinuclear region (C), but when they were treated with NMMA, some stress fibers became apparent in cytoplasm and a prominent dense band of fluorescence (arrowheads) was evident at cell periphery (D). Original magnification, ×400. Journal of Vascular Surgery  , DOI: ( /S (97) ) Copyright © 1997 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

12 Fig. 5 Effect of glucose concentration on endothelial cell migration. Elevated glucose in culture medium inhibited cell migration as determined by cell number (A), distance migrated (B), or surface area covered (C). Inhibition was maximal at 16.6 mmol/L glucose and was not the result of any physical effect of increased glucose, because L-glucose at 27.7 mmol/L did not influence any parameters of migration. Journal of Vascular Surgery  , DOI: ( /S (97) ) Copyright © 1997 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

13 Fig. 5 Effect of glucose concentration on endothelial cell migration. Elevated glucose in culture medium inhibited cell migration as determined by cell number (A), distance migrated (B), or surface area covered (C). Inhibition was maximal at 16.6 mmol/L glucose and was not the result of any physical effect of increased glucose, because L-glucose at 27.7 mmol/L did not influence any parameters of migration. Journal of Vascular Surgery  , DOI: ( /S (97) ) Copyright © 1997 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

14 Fig. 5 Effect of glucose concentration on endothelial cell migration. Elevated glucose in culture medium inhibited cell migration as determined by cell number (A), distance migrated (B), or surface area covered (C). Inhibition was maximal at 16.6 mmol/L glucose and was not the result of any physical effect of increased glucose, because L-glucose at 27.7 mmol/L did not influence any parameters of migration. Journal of Vascular Surgery  , DOI: ( /S (97) ) Copyright © 1997 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

15 Fig. 6 Regulation of endothelial cell migration by modulators of NO. Addition to cultures of NMMA (0.15 mmol/L) prevented inhibitory effect of elevated glucose (A), whereas addition of S-nitrosylated penicillamine inhibited normal migration as evidenced by total number of migrating cells (B). Total surface area and distance migrated followed the same pattern as total number of cells. Journal of Vascular Surgery  , DOI: ( /S (97) ) Copyright © 1997 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

16 Fig. 6 Regulation of endothelial cell migration by modulators of NO. Addition to cultures of NMMA (0.15 mmol/L) prevented inhibitory effect of elevated glucose (A), whereas addition of S-nitrosylated penicillamine inhibited normal migration as evidenced by total number of migrating cells (B). Total surface area and distance migrated followed the same pattern as total number of cells. Journal of Vascular Surgery  , DOI: ( /S (97) ) Copyright © 1997 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

17 Fig. 7 Effect of glucose on endothelial cell proliferation. Elevated glucose (27.7 mmol/L) increased cell proliferation in the presence of 2% FBS, had no effect at 5 and 10% FBS, and inhibited proliferation at 20% FBS. Addition of NMMA (0.15 mmol/L) had no effect on cell proliferation. *Statistically significant difference; p < 0.05. Journal of Vascular Surgery  , DOI: ( /S (97) ) Copyright © 1997 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

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