1. The coronary artery bypass conduit: I
The coronary artery bypass conduit: I. Intraoperative endothelial injury and its implication on graft patency 
Hemant S. Thatte, PhD, Shukri F. Khuri, MD 
The Annals of Thoracic Surgery 
Volume 72, Issue 6, Pages S2245-S2252 (December 2001)
DOI: /S (01)

2. Fig 1
Stage 1 (<1 month after CABG surgery): explantation of saphenous vein leads to denudation and damage to the endothelium. Platelets (brown filled circles), neutrophils (pink filled squiggles [resembling “approximate” signs]), monocytes (green filled circles), and fibrin (light purple wavy lines) are recruited on the exposed basement membrane (BM) and extracellular matrix (EM), resulting in decrease in release of anticoagulant and vasorelaxant factors (red downward arrow) and increased secretion of procoagulant and vasoconstrictor effectors (red upward arrows), leading to thrombogenesis, endothelial cell (EC) activation, and inflammation. Stage 2 (>1 month after surgery): activation, inflammation, and aggregation of EC, platelets, and recruitment of leukocytes induces intimal hyperplasia by the proliferation of smooth muscle cells (SMC). Stage 3 (generally >3 years after surgery): monocytes transformed into macrophages, migrate to the subendothelial layers, accumulate lipid particles, and become foam cells. Simultaneously, SMC migrate and proliferate into the lumen, entrapping foam cells, cellular debris (brown wavy lines) and recruited blood cells forming a plaque. Stages 1 and 2 result in EC failure, loss of vasomotor function, and ultimately stenosis and graft failure.
The Annals of Thoracic Surgery  , S2245-S2252DOI: ( /S (01) )

3. Fig 2
Cell viability assay in intact human saphenous vein. A freshly segmented vein was labeled with calcein and ethidium homodimer in Hank’s balanced salt solution. (A) Green cellular fluorescence indicates cell viability. (B) Red nuclear fluorescence shows compromised or dead cells. The smooth muscle cell layer in the intimal region of the vein is identifiable at ∼100 μm and the vein lumen becomes visible at ∼150 μm. To differentiate between living and dead cells, the endothelial cells were made permeable with 0.1% triton before labeling as shown in part B. Figure 2 demonstrates the ability to identify the lumen and endothelium, as well as to differentiate between living and dead cells in an ex vivo, intact vein using multiphoton microscopy.
The Annals of Thoracic Surgery  , S2245-S2252DOI: ( /S (01) )

4. Fig 3
Real-time imaging of nitric oxide generated in saphenous vein segments using multiphoton microscopy. The vein segments were incubated with nitric oxide indicator dye DAF-2/DA in Hank’s balanced salt solution for 1 hour at 37oC and mounted on the microscope stage. After identifying the vein lumen by XYZ scanning, microscope was slightly defocused allowing for larger endothelial area for quantification of nitric oxide. By this maneuver, a larger endothelial cell area was available for quantitation of nitric oxide. eNOS was activated with bradykinin (10 μmol/L) and the temporal increase in nitric oxide–mediated fluorescence was measured. (A) Representative image of eNOS activity before activation. (B) The same vein segment imaged 10 minutes after eNOS activation. A 2.5- to 3-fold increase in fluorescence due to the production of nitric oxide was observed.
The Annals of Thoracic Surgery  , S2245-S2252DOI: ( /S (01) )

5. Fig 4
Temporal changes in human saphenous vein eNOS activity. The vein segments were incubated with DAF-2/DA and processed for endothelial nitric oxide generation. The integrated DAF-2 fluorescence intensity of endothelial region of each vein stored at various time points was measured after 10 minutes of treatment with bradykinin (10 μmol/L) and normalized to the fluorescence intensity measured before the drug treatment. Six independent vein preparations were examined. Changes in fluorescence intensity between basal and bradykinin-treated vessels decreased significantly with time of storage and between various time points. Bars represent mean ± SEM. p = by analysis of variance for 10 minutes after bradykinin treatment over time; * p = 0.026, prebradykinin versus postbradykinin at 60-minute value; ** p = 0.017, postbradykinin 60-minute versus 120-minute value; ***p = 0.002, postbradykinin 60-minute versus 240-minute value. (Reproduced from Ref. 22 by permission).
The Annals of Thoracic Surgery  , S2245-S2252DOI: ( /S (01) )

6. Fig 5
pH-Dependent viability of vein endothelium. Human saphenous vein segments were stored in Hank’s balanced salt solution at various pH for 60 minutes at 21°C and were then labeled with calcein/ethidium homodimer. Green cellular fluorescence indicates cell viability and red nuclear fluorescence shows compromised or dead cells. (A) Cell viability was well preserved in veins stored at pH 7.4. (B) Significant cell death can be seen in vessels stored at pH 6. (C) Combination of living and dead cells are visible in veins stored at pH 8.0. SMC = smooth muscle cells. (Reproduced from Ref. 22 by permission).
The Annals of Thoracic Surgery  , S2245-S2252DOI: ( /S (01) )

7. Fig 6
Distension-dependent changes in vessel viability. Saphenous vein and internal mammary artery segment were labeled with calcein/ethidium homodimer. Green cellular fluorescence indicates cell viability; red nuclear fluorescence shows compromised or dead cells. (A) Saphenous vein segment: living endothelium and intimal smooth muscle cells (SMC). (B) Distended saphenous vein segment: denuded and damaged endothelial and SMC. (C) Internal mammary artery before anastomosis: living endothelium and SMC regions. Note the differences in endothelial architecture of saphenous vein (A) and internal mammary artery (B).
The Annals of Thoracic Surgery  , S2245-S2252DOI: ( /S (01) )

8. Fig 7
Distension mediated inactivation of eNOS activity in human saphenous veins. The control (undistended) and distended vein segments were incubated with DAF-2/DA and processed for endothelial nitric oxide generation. The integrated DAF-2 fluorescence intensity of endothelial region of each vein stored at various time points was measured after 10 minutes of treatment with bradykinin (10 μmol/L) and normalized to fluorescence intensity measured before drug treatment. Three independent vein preparations were examined. Increase in fluorescence intensity due to nitric oxide generation was significantly greater in undistended vein compared with distended vein (p < 0.01). Bars represent mean ± SEM.
The Annals of Thoracic Surgery  , S2245-S2252DOI: ( /S (01) )