Role of thrombin in endothelial cell monolayer repair in vitro

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Presentation transcript:

Role of thrombin in endothelial cell monolayer repair in vitro Paul J. DiMuzio, MD, Kerri J. Pratt, PhD, Pauline K. Park, MD, R.Anthony Carabasi, MD Journal of Vascular Surgery Volume 20, Issue 4, Pages 621-628 (October 1994) DOI: 10.1016/0741-5214(94)90287-9 Copyright © 1994 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 1 Effect of thrombin on monolayer repair. A, After 3 days, 1 to 8 U/ml thrombin inhibited human iliac artery endothelial cell repair distance in dose-related manner (*p < 0.02). B, Thrombin dose-response curve (day 3) reveals high degree of correlation between inhibition of monolayer repair and increasing thrombin dose (R = 0.99). Journal of Vascular Surgery 1994 20, 621-628DOI: (10.1016/0741-5214(94)90287-9) Copyright © 1994 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 1 Effect of thrombin on monolayer repair. A, After 3 days, 1 to 8 U/ml thrombin inhibited human iliac artery endothelial cell repair distance in dose-related manner (*p < 0.02). B, Thrombin dose-response curve (day 3) reveals high degree of correlation between inhibition of monolayer repair and increasing thrombin dose (R = 0.99). Journal of Vascular Surgery 1994 20, 621-628DOI: (10.1016/0741-5214(94)90287-9) Copyright © 1994 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 2 Reversibility of thrombin's effect. Average repair rate (distance traveled/time) for 3 days was less for thrombin-treated cells (0.21 ± 0.01 mm/d) compared with that of the control group (0.49 ± 0.01 mm/d) (*p < 0.001). After removal of thrombin at day 3, increase in repair rate was seen (0.41 ± 0.01 mm/d) compared with that seen before break (0.21 ± 0.01 mm/d) (**p <0.001). Journal of Vascular Surgery 1994 20, 621-628DOI: (10.1016/0741-5214(94)90287-9) Copyright © 1994 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 3 Interaction between thrombin and fetal bovine serum. A, Monolayer repair after 3 days in 12% serum was significantly increased compared with experiments in 4% serum (*p < 0.001). B, Normalization of these curves for each serum concentration reveals that inhibitory effect of thrombin in 12% serum was significantly less than that seen at 4% (*p < 0.001 for both 1 and 4 U/ml). Journal of Vascular Surgery 1994 20, 621-628DOI: (10.1016/0741-5214(94)90287-9) Copyright © 1994 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 3 Interaction between thrombin and fetal bovine serum. A, Monolayer repair after 3 days in 12% serum was significantly increased compared with experiments in 4% serum (*p < 0.001). B, Normalization of these curves for each serum concentration reveals that inhibitory effect of thrombin in 12% serum was significantly less than that seen at 4% (*p < 0.001 for both 1 and 4 U/ml). Journal of Vascular Surgery 1994 20, 621-628DOI: (10.1016/0741-5214(94)90287-9) Copyright © 1994 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 4 Interaction between thrombin and endothelial cell growth supplement. A, Addition of endothelial cell growth supplement (40 μg/ml) significantly stimulated monolayer repair after 3 days (*p < 0.001). B, Normalization of these curves for each serum concentration fails to demonstrate any difference in effect of thrombin in presence or absence of endothelial cell growth supplement (p > 0.05). Journal of Vascular Surgery 1994 20, 621-628DOI: (10.1016/0741-5214(94)90287-9) Copyright © 1994 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 4 Interaction between thrombin and endothelial cell growth supplement. A, Addition of endothelial cell growth supplement (40 μg/ml) significantly stimulated monolayer repair after 3 days (*p < 0.001). B, Normalization of these curves for each serum concentration fails to demonstrate any difference in effect of thrombin in presence or absence of endothelial cell growth supplement (p > 0.05). Journal of Vascular Surgery 1994 20, 621-628DOI: (10.1016/0741-5214(94)90287-9) Copyright © 1994 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 5 Effect of proteolytically inactive di-isopropylfluorophosphate- thrombin. During 2-day repair experiment, di-isopropylfluorophosphate-thrombin (4 U/ml) failed to produce any significant alteration in wound healing (p > 0.05). Human α-thrombin (4 U/ml) with intact catalytic site inhibited monolayer repair (*p < 0.001). Journal of Vascular Surgery 1994 20, 621-628DOI: (10.1016/0741-5214(94)90287-9) Copyright © 1994 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 6 Effect of thrombin on cell proliferation. A, In 12% serum 4 to 8 U/ml thrombin inhibited log and plateau phases of human iliac artery endothelial cell growth (p < 0.001 at days 8 and 13). B, In 4% serum, however, as little as 0.5 U/ml thrombin inhibited cell growth (p < 0.01 at days 8 and 13). Journal of Vascular Surgery 1994 20, 621-628DOI: (10.1016/0741-5214(94)90287-9) Copyright © 1994 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 6 Effect of thrombin on cell proliferation. A, In 12% serum 4 to 8 U/ml thrombin inhibited log and plateau phases of human iliac artery endothelial cell growth (p < 0.001 at days 8 and 13). B, In 4% serum, however, as little as 0.5 U/ml thrombin inhibited cell growth (p < 0.01 at days 8 and 13). Journal of Vascular Surgery 1994 20, 621-628DOI: (10.1016/0741-5214(94)90287-9) Copyright © 1994 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 7 Proliferation of wounded monolayers. Bromodeoxyuridine uptake is increased in control cells located at wound edge and wound background compared with that in unwounded control monolayers (*p < 0.02). Thrombin (4 U/ml) inhibited bromodeoxyuridine uptake in cells proliferating at the wound edge compared with that in control group (**p < 0.001). It prevented any significant increase in proliferation noted in wound background. Thrombin did not alter proliferation of unwounded monolayers (p > 0.05). Journal of Vascular Surgery 1994 20, 621-628DOI: (10.1016/0741-5214(94)90287-9) Copyright © 1994 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 8 Effect of thrombin on human iliac artery endothelial cell versus human umbilical vein endothelial cell growth. After 6 days of growth percent change in cell count is plotted for each thrombin concentration. Thrombin inhibited human iliac artery endothelial cell growth (*p < 0.05), whereas it stimulated growth of human umbilical vein endothelial cell (*p < 0.05). Journal of Vascular Surgery 1994 20, 621-628DOI: (10.1016/0741-5214(94)90287-9) Copyright © 1994 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions