-- Laki J. Rousou, MS, MD et. al -- Boston, Massachusetts. -- Ann Thorac Surg 2009;87:62 70. Presented by: Int. 黃柏翔 Date: 2009 /02 / 25 Patient outcome after CABG surgery greatly influenced by patency rates of saphenous vein (SV) grafts. Endothelium. Physiologic barrier between blood components and sub-endothelium. Injury Initiation site of later-stage atheromas and graft failure. Harvesting SV grafts: Prevention of injury. Decrease surgical manipulation. Different approaches to harvesting SV grafts. Traditional open SV harvest (OSVH). -- Long continuous incision from groin to ankle with minimal manipulation of SV graft. Endoscopic harvest (ESVH). -- Decrease lower extremity morbidity. PREVENT-IV study (Project of Ex Vivo Vein Graft Engineering Via Transfection) ESVH an independent predictor of decreased SV patency at 1 year. Main objective of this study: -- Compare viability and functionality of SV endothelium after OSVH vs. ESVH.
Prospectively identified 10 male patients, 10 aged 60 to 81 years scheduled for elective CABG surgery at VA Boston Healthcare System from 2007/07 ~ 2007/10. Each patient underwent both ESVH for proximal portion and OSVH for distal portion of SV. ESVH. CO2 insufflation for visualization/dissection of tissue. Side branches cauterized with bipolar cautery. Endoscopic portion of SV graft excised with stab incision at groin. Maintained close sampling proximity. OSVH. Standard no touch technique. Both harvest techniques performed by one physician s assistant with over 110 ESVH experiences. 1 cm portion of both OSVH and ESVH were transported back to the multiphoton microscopy (MPM) lab immediately. MPM a sensitive instrument to measuring endothelial viability and functionality in real time. Endothelial cell structure / viability. Fluorescence-based supervitality Live-Dead assay. -- Calcein AM / Ethidium homodimer. Viable cells: Calcein AM ester (non-fluorescent) esterase activity Fluorescent Calcein (green). Structural damaged cells: Permiability to Ethidium homodimer (red).
Intracellular Ca mobilization / NO generation. Calcium-sensitive orange dye / NO-specific diaminofluorescein dye. Measure resting Ca levels / basal activity of endothelial nitric oxide synthase (enos). Add Bradykinin Stimulation of Ca mobilization, NO generation. Caveolin 3, enos, Cadherin, vwf. Immunofluorescence. Primary protein Ab Secondary fluorscein-conjugated Ab. Western Blotting. Electrophoresis. The above 3 methods where documented by both imaging and quantitative analysis. Endothelial cell structure / viability. Fluorescence-based supervitality Live-Dead assay. -- Calcein AM / Ethidium homodimer. Viable cells: Calcein AM ester (non-fluorescent) esterase activity Fluorescent calcein (green). Structural damaged cells: Permiability to ethidium homodimer (red).
Esterase activity Intracellular Ca mobilization / NO generation. Calcium-sensitive orange dye / NO-specific diaminofluorescein dye. Measure resting Ca levels / basal activity of endothelial nitric oxide synthase (enos). Add Bradykinin Stimulation of Ca mobilization, NO generation. Ca mobilization NO production Caveolin 3, enos, Cadherin, vwf. Immunofluorescence. Primary protein Ab Secondary fluorscein-conjugated Ab. Western Blot. Electrophoresis. Immunofluorescence
Western blot Immunofluorescence Western blot Esterase activity in endothelium. OSVH > ESVH. Higher cellular metabolic activity, viability, less membrane damage in OSVH.
Ca mobilization in endothelium. OSVH > ESVH. Important upstream 2 nd messenger in signal conduction, including: NO production, endothelial-dependent vasomotor function. NO production in endothelium. OSVH > ESVH. Impaired NO secretion leads to platelet aggregation, recruitment, aggregation and thrombus formation. NO secretion induces smooth muscle relaxation, prevents neutrophil-endothelial adhesion, atherosclerosis. Caveolin & enos in endothelium. OSVH > ESVH. Caveolin: docking protein in cell membrane required for activation of enos and production of NO. enos: directly involved in production of NO. Significantly impacts graft patency and patient outcome. Conclusion: ESVH has detrimental effect on SV endothelium, which leads to decreased graft patency and worse patient outcomes. Limitations. MPM too sensitive?! Endothelial dysfunction reversible?! In vitro study requires correlation with clinical sequelae.