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Wai-ki Yiu, MBBS, MRCS, Stephen W. K. Cheng, MS, FRCS, Bauer E

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Presentation on theme: "Wai-ki Yiu, MBBS, MRCS, Stephen W. K. Cheng, MS, FRCS, Bauer E"— Presentation transcript:

1 Direct comparison of endothelial cell and smooth muscle cell response to supercooling and rewarming 
Wai-ki Yiu, MBBS, MRCS, Stephen W.K. Cheng, MS, FRCS, Bauer E. Sumpio, MD, PhD  Journal of Vascular Surgery  Volume 46, Issue 3, Pages e2 (September 2007) DOI: /j.jvs Copyright © 2007 The Society for Vascular Surgery Terms and Conditions

2 Fig 1 Apoptosis of (A) endothelial cells (ECs) and (B) smooth muscle cells (SMCs) after supercooling and rewarming as evaluated by terminal deoxynucleotide transferase-mediated deoxy uridine triphosphate nick-end labeling (TUNEL) assay. The percentage of positive TUNEL cells is indicated on the y axis and the rewarming time is indicated on the x axis. The clear bar represents 0-second cooling group, the diagonal bar represents 60-second cooling group, and the dotted bar represents 120-second cooling group. A, Apoptosis of ECs, as indicated by the TUNEL-positive cells, increased with supercooling and rewarming time (*P < .05 compared with 0 hour rewarming and same cooling time; αP < .05 compared with 6 hours rewarming and same cooling time; βP < .05 compared with 0 seconds cooling, followed by same hour rewarming; γP < .05 compared with 60 seconds cooling, followed by same hour rewarming). B, Apoptosis of SMCs as indicated by the TUNEL-positive cells increased with supercooling and rewarming time (*P < .05 compared with 0 hours rewarming with same cooling time; βP < .05 compared with 6 hours rewarming with same cooling time; αP < .05 compared with 12 hours rewarming with the same cooling time; μP < .05 compared with 0 seconds cooling with same rewarming hours; †P < .05 compared with 60 seconds cooling with same rewarming hours). Data are presented with the standard error of the mean. Journal of Vascular Surgery  , e2DOI: ( /j.jvs ) Copyright © 2007 The Society for Vascular Surgery Terms and Conditions

3 Fig 2 Western blots of protein kinase B (AKT) phosphorylation after supercooling and rewarming in (A) endothelial cells (EC) and (B) smooth muscle cells (SMC). Representative immunoblots are of AKT phosphorylation after 0, 60, and 120 seconds supercooling, followed by 0, 6, 12, and 24 hours rewarming. The upper immunoblots represent the phosphorylation of AKT (Ser 473) (pAkt) and the lower immunoblots represent the loading control using anti-total Akt antibody (tAkt). Data are presented with the standard error of the mean. A, Significant activation was noted after 120 seconds cooling, followed by 24 hours rewarming (*P < .05 compared with 0 hours rewarming). B, Significant activation was noted after 60 seconds cooling, followed by 24 hours rewarming (*P < .05 compared with 0 hours rewarming). Journal of Vascular Surgery  , e2DOI: ( /j.jvs ) Copyright © 2007 The Society for Vascular Surgery Terms and Conditions

4 Fig 3 Proliferation of (A) vascular endothelial cells (EC) and (B) vascular smooth muscle cells (SMC) after supercooling and re-warming as evaluated by 5′-bromo-2′-deoxyuridine (BrdU) assay. The percentage of BrdU labeling is indicated on the y axis and the rewarming time is indicated on the x axis. Data are presented with the standard error of the mean. A, Significant decrease of proliferation was noted after 6, 12, and 24 hours rewarming in the noncooling group, and 60 and 120 seconds cooling without rewarming compared with the noncooling and nonrewarming group (*P < .05 compared with 0 hours rewarming and same cooling condition; †P < .05 compared with 0 seconds cooling and same rewarming time; αP < .05 compared with 12 hours rewarming and same cooling condition). B, No significant changes in proliferation rate were noted except at 120 seconds of cooling, followed by 6 and 24 hours of re-warming compared with the same cooling condition but without re-warming (*P < .05 compared with 0 hour rewarming and the same cooling condition). Journal of Vascular Surgery  , e2DOI: ( /j.jvs ) Copyright © 2007 The Society for Vascular Surgery Terms and Conditions

5 Fig 4 Western blot of P70 S6 kinase phosphorylation after supercooling and rewarming in (A) endothelial cells (EC) and (B) smooth muscle cells (SMC). Representative blots of P70 S6 kinase phosphorylation after 0, 60, and 120 seconds cooling, followed by 0, 6, 12, and 24 hours rewarming. The top immunoblots were performed with antiphospho-P70 S6 kinase antibody (Thr 421/Ser 424) and the lower immunoblots were performed with antitotal P70 S6 kinase as a control of loading. Densitometeric analysis of P70 S6 kinase phosphorylation after normalization with loading control is shown. The fold of activation is indicated on the y axis and the rewarming hours are indicated on the x-axis. Data are presented with the standard error of the mean. A, Significant activation was noted after 60 seconds cooling, followed by 12 hours rewarming (*P < .05 compared with 0 hours rewarming group under the same cooling condition). B, The P70S6 kinase activity gradually decreased with significant drop at 0 seconds, followed by 6, 12, and 24 hours rewarming and 60 seconds cooling, followed by 12 hours rewarming (*P < .05 compared with 0 hours rewarming group under the same cooling condition). Journal of Vascular Surgery  , e2DOI: ( /j.jvs ) Copyright © 2007 The Society for Vascular Surgery Terms and Conditions

6 Fig 5 Western blot of P44/42 mitogen-activated protein kinase (MAPK) phosphorylation after supercooling and rewarming in (A) endothelial cells (EC) and (B) smooth muscle cells (SMC). Shown are the representative blots of P44/42 MAP kinase after 0, 60, and 120 seconds cooling, followed by 0, 6, 12, and 24 hours rewarming. The top immunoblots were performed with anti-phospho-P44/42 MAP kinase antibody (Tyr 202/Tyr 204) and the low immunoblots were performed with anti-P44/42 MAP kinase antibody. The densitometeric analysis of P44/42 MAP kinase phosphorylation after normalization with the loading control is shown. The y axis represents the fold of activation of P44/42 MAPK and x-axis represents the rewarming hours. A, Increased EC activation was observed with supercooling and rewarming, with a significant activation at 120 seconds cooling, followed by 24 hours rewarming (αP < .05 compared with 6 hours rewarming under same cooling condition). B, The activity of P44/42 MAP kinase phosphorylation decreased gradually with time, with a significant drop at 0 seconds cooling, followed by 6 and 12 hours rewarming and 60 seconds cooling followed by 12 hours rewarming (*P < .05 compared with 0 hours rewarming and the same cooling time). Journal of Vascular Surgery  , e2DOI: ( /j.jvs ) Copyright © 2007 The Society for Vascular Surgery Terms and Conditions

7 Fig E1, online only Diagram shows the design of cryostage. The flow channels inside the cryostage were created symmetrically so that the same temperature would be delivered to each of the six wells of the culture plate. Journal of Vascular Surgery  , e2DOI: ( /j.jvs ) Copyright © 2007 The Society for Vascular Surgery Terms and Conditions

8 Fig E2, online only Cooling curve of medium inside the each well of culture plate. The x axis represents the time on the cryostage. The y axis represents the temperature changes of medium inside each well. The curve demonstrates the close similarity of temperature changes in each well. Journal of Vascular Surgery  , e2DOI: ( /j.jvs ) Copyright © 2007 The Society for Vascular Surgery Terms and Conditions

9 Fig E3, online only Temperature changes of the PolarCath during cryoplasty. The x axis represents the time of cryoplasty, and the y axis represents the temperature changes inside the balloon. The temperature dropped to −10°C at 30 seconds. This is compatible with our cooling curve using 1 mL of medium in culture plate put on the cryostage set at −21°C. Journal of Vascular Surgery  , e2DOI: ( /j.jvs ) Copyright © 2007 The Society for Vascular Surgery Terms and Conditions

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