Rho and basic fibroblast growth factor involvement in centrosome redistribution and actin microfilament remodeling during early endothelial wound repair

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Rho and basic fibroblast growth factor involvement in centrosome redistribution and actin microfilament remodeling during early endothelial wound repair Tsu-Yee Joseph Lee, PhD, Avrum I. Gotlieb, MD Journal of Vascular Surgery Volume 35, Issue 6, Pages 1242-1252 (June 2002) DOI: 10.1067/mva.2002.123094 Copyright © 2002 Society for Vascular Surgery and The American Association for Vascular Surgery Terms and Conditions

Fig. 1 Percentage of cells at wound edge with specific microfilament orientation (A) or specific centrosome position (B) in response to exogenous bFGF or exoenzyme C3 3 hours after wounding. bFGF 10 ng/mL significantly decreased percentage of cells exhibiting central microfilaments parallel to wound edge and significantly increased percentage of cells exhibiting central microfilaments perpendicular to wound edge as compared with control (A). C3 2 μg/mL caused significant central microfilament loss (A) and significantly decreased centrosome distribution towards wound (B) as compared with control. Coincubation of 10 or 15 ng/mL bFGF with 2 μg/mL C3 did not reverse C3-induced central microfilament loss (A) but did restore centrosome distribution towards wound back to control values. Differences in central microfilament orientation or in centrosome position between control and treatment groups were significant at *P <.05. Journal of Vascular Surgery 2002 35, 1242-1252DOI: (10.1067/mva.2002.123094) Copyright © 2002 Society for Vascular Surgery and The American Association for Vascular Surgery Terms and Conditions

Fig. 2 Confocal photomicrographs of control (A), bFGF-treated (B), C3-treated (C), or bFGF with C3-treated (D) endothelial cells at wound edge 3 hours after wounding double-stained for F-actin and α-tubulin. Wound is at top. A, Control cells had central microfilaments parallel to wound edge. B, bFGF-treated (10 ng/mL) cells exhibited central microfilaments perpendicular to wound edge. C, C3-treated (2 μg/mL) cells had severe central microfilament loss, decreased cell lengths, reduced lamellipodia extrusions, and lateral gaps between adjacent cells. Coincubation of 10 ng/mL bFGF with 2 μg/mL C3 produced similar effects as C3 alone, except cell lengths appeared to be longer. Bar, 25 μm. Journal of Vascular Surgery 2002 35, 1242-1252DOI: (10.1067/mva.2002.123094) Copyright © 2002 Society for Vascular Surgery and The American Association for Vascular Surgery Terms and Conditions

Fig. 3 Confocal photomicrographs of control (A,B) and 2 μg/mL C3-treated (C,D) endothelial cells at wound edge 3 hours after wounding double-stained for F-actin (red) and phosphotyrosine (green;A,C) or F-actin (red) and paxillin (green;B,D). Wound is at top. In control cells, phosphotyrosine (A) and paxillin (B) localized at ends of central microfilaments parallel to wound edge and at tips of filopodia. In C3-treated cells, central microfilament loss was associated with reduction of both phosphotyrosine (C) and paxillin (D). However, phosphotyrosine was still seen at ends of filopodia extending into wound and laterally (C). Bar, 25 μm. Journal of Vascular Surgery 2002 35, 1242-1252DOI: (10.1067/mva.2002.123094) Copyright © 2002 Society for Vascular Surgery and The American Association for Vascular Surgery Terms and Conditions

Fig. 4 Confocal photomicrographs of control (A), bFGF-treated (B), C3-treated (C), or bFGF with C3-treated (D) endothelial cells at wound edge 3 hours after wounding stained for α-tubulin (see Fig 1, B). Wound is at top. Control cells (A) and 10 ng/mL bFGF-treated cells (B) had centrosome redistribution to front of cell towards wound. C, C3 treatment (2 μg/mL) did not result in microtubule loss but did inhibit centrosome redistribution towards wound. D, Coincubation of 10 ng/mL bFGF with 2 μg/mL C3 restored centrosome redistribution towards wound and increased cell lengths. Centrosome is identified as brightly stained amorphous area of tubulin (arrow) near nucleus (N). Bar, 25 μm. Journal of Vascular Surgery 2002 35, 1242-1252DOI: (10.1067/mva.2002.123094) Copyright © 2002 Society for Vascular Surgery and The American Association for Vascular Surgery Terms and Conditions

Fig. 5 Confocal magnified photomicrographs of control endothelial cells in confluent monolayer (A) and at wound edge (B) and of endothelial cells at wound edge treated with 2 μg/mL C3 (C) or cotreated with 10 ng/mL bFGF and 2 μg/mL C3 (D) 3 hours after wounding. Endothelial cells were double-stained for RhoA (red) and α-tubulin (green). Wound is at top. A, In confluent monolayer, punctate RhoA staining was concentrated with centrosome and alongside microtubules. B, After wounding, RhoA was concentrated with redistributed centrosome and alongside some microtubules that radiated outwards from centrosome. C, Addition of C3 resulted in RhoA localization with less defined and slightly fragmented microtubules and with nonredistributed centrosome. Note that C3 resulted in less RhoA staining in areas devoid of polymerized microtubules (C,arrow) when compared with control wound edge cells but that RhoA was seen in areas of highly disrupted microtubules (C, arrowhead). D, Coincubation of bFGF with C3 restored centrosome redistribution towards wound. RhoA localized with redistributed centrosome and along microtubules. Centrosome (c) is identified as brightly stained amorphous area of α-tubulin near nucleus (N). Bars, 5 μm. Journal of Vascular Surgery 2002 35, 1242-1252DOI: (10.1067/mva.2002.123094) Copyright © 2002 Society for Vascular Surgery and The American Association for Vascular Surgery Terms and Conditions

Fig. 6 Confocal photomicrographs of control (A), C3-treated (B), or bFGF with C3-treated (C) endothelial cells at wound edge 3 hours after wounding double-stained for F-actin (red) and acetylated α-tubulin (green). Wound is at top. A, Control cells exhibited long acetylated α-tubulin processes extending from centrosome to posterior cell border (arrowhead). B, C3 treatment (2 μg/mL) resulted in loss of long acetylated α-tubulin processes associated with decreased cell elongation and inhibition of centrosome redistribution. C, Coincubation of 10 ng/mL bFGF with 2 μg/mL C3 restored long acetylated α-tubulin processes extending from centrosome to posterior cell border (arrowhead) and increased cell elongation and centrosome redistribution. Acetylated α-tubulin tended to concentrate with centrosome near nucleus (N). Bar, 25 μm. Journal of Vascular Surgery 2002 35, 1242-1252DOI: (10.1067/mva.2002.123094) Copyright © 2002 Society for Vascular Surgery and The American Association for Vascular Surgery Terms and Conditions