Vein Graft Harvesting Induces Inflammation and Impairs Vessel Reactivity Kazuhiro Hinokiyama, MD, Guro Valen, MD, PhD, Shinichi Tokuno, MD, PhD, Jenny.

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Vein Graft Harvesting Induces Inflammation and Impairs Vessel Reactivity Kazuhiro Hinokiyama, MD, Guro Valen, MD, PhD, Shinichi Tokuno, MD, PhD, Jenny Bäcklund Vedin, MD, PhD, Jarle Vaage, MD, PhD The Annals of Thoracic Surgery Volume 82, Issue 4, Pages 1458-1464 (October 2006) DOI: 10.1016/j.athoracsur.2006.05.038 Copyright © 2006 The Society of Thoracic Surgeons Terms and Conditions

Fig 1 Representative electrophoretic mobility shift assays of nuclear protein extracted from saphenous veins of patients at the start of surgical preparation (V1) and at the time of completing the last proximal anastomosis to the heart (V2) during open heart surgery. The protein extracts were incubated with a radio-labelled probe containing the nuclear factor-κ B (NFκB) motif. Samples from 6 different patients are shown. Surgical handling resulted in increased band density in most patients, indicating NFκB activation. The band identity was verified with cold probe competition in 50-fold or 100-fold excess (+50cp, +100cp), with supershift analysis with a NFκB p50 and a p65 antibody (+p50, +p65), and with non-sense competition with a cold AP-1 probe (+AP-1, lower panel). The Annals of Thoracic Surgery 2006 82, 1458-1464DOI: (10.1016/j.athoracsur.2006.05.038) Copyright © 2006 The Society of Thoracic Surgeons Terms and Conditions

Fig 2 (A) A representative polyacrylamide gel with radio-labelled reverse transcription-polymerase chain reaction (RT-PCR) products from saphenous vein grafts of 1 patient with stable angina undergoing coronary artery bypass grafting. Samples were taken at the start of surgical handling (V1) and after surgical handling (V2). Histone H3 (H3, 215 bp) was used as a reference gene to evaluate the relative amount of test genes, which were tumor necrosis factor-α (TNF-α, 444 bp; interleukin 1β (IL-1β, 464 bp), E-selectin (CD64E, 255 bp), intercellular adhesion molecule-1 (ICAM-1, 237 bp), endothelin-1 (ET-1, 737 bp), inducible nitric oxide synthase (NOS2, 532 bp), and endothelial NOS (NOS3, 725 bp). The ratio of optical density of the test gene band and H3 were used as a basis for the calculation shown in B. (B) Gene expression changes in vein grafts (n = 9). When the test gene band optical density of the PCR products of all 9 patients were measured and divided by the density of the housekeeping gene H3 band, gene expression TNF-α, IL-1β, CD62E, and ICAM-1 were significantly increased by surgical handling. ET-1, NOS2, and NOS3 were not induced in the vessel wall by routine surgical procedures in this time frame. Relative messenger RNA expression is shown as box plots with median, and the whiskers show the range. White bars = start of surgical handling (V1); gray bars = after surgical handling (V2). The Annals of Thoracic Surgery 2006 82, 1458-1464DOI: (10.1016/j.athoracsur.2006.05.038) Copyright © 2006 The Society of Thoracic Surgeons Terms and Conditions

Fig 3 A representative morphology of the graft material taken at the start of surgical preparation (a), and after harvesting, handling, and testing for leakage (b). Samples from one patient are shown. Note the flaccid and distended appearance after surgical preparation. When immunostaining with an antibody against the endothelial marker von Willebrand factor was used, a brown monolayer of cells could be seen at the start of graft harvesting (c), but this was partially disrupted at the time of completing the proximal anastomoses to the heart (d). Counterstaining was performed with hematoxylin. For quantification of loss of endothelium in all investigated patients, see Results. Original magnification × 100 (a, b), original magnification × 400 (c, d). The Annals of Thoracic Surgery 2006 82, 1458-1464DOI: (10.1016/j.athoracsur.2006.05.038) Copyright © 2006 The Society of Thoracic Surgeons Terms and Conditions

Fig 4 Rings of human saphenous veins were collected at the start of preparation and after the proximal anastomoses were performed. The rings were mounted in organ baths, where dose-dependent contractions to phenylephrine (top) and endothelin-1 (bottom) were observed. In the vein grafts sampled at the start of surgical handling, a dose-dependent contraction to both contractile agents was observed (a and b). However, almost all contraction was lost during preparation and handling of the graft. V1 = start of graft harvesting, V2 = after surgical handling. Data are mean ± SD, n = 9. *Denotes p < 0.05 when comparing before and after. The Annals of Thoracic Surgery 2006 82, 1458-1464DOI: (10.1016/j.athoracsur.2006.05.038) Copyright © 2006 The Society of Thoracic Surgeons Terms and Conditions