Minimally Invasive Saphenous Vein Harvesting: Effects on Endothelial and Smooth Muscle Function

Minimally Invasive Saphenous Vein Harvesting: Effects on Endothelial and Smooth Muscle Function Edward A. Black, FRCS, Tomasz J. Guzik, MD, Nick E. J. West, MRCP, Karen Campbell, RN, Ravi Pillai, FRCS, Chandi Ratnatunga, FRCS, and Keith M. Channon, MD, MRCP Oxford Heart Centre and Department of Cardiovascular Medicine, John Radcliffe Hospital, University of Oxford, Oxford, United Kingdom Background. The long saphenous vein remains the commonest conduit used in coronary artery bypass grafting procedures. Surgical trauma during vein harvesting can cause endothelial and smooth muscle injury that has important implications for vein graft longevity. Minimally invasive vein harvesting is advocated to reduce wound morbidity. However, the functional consequences of increased handling and traction, with potentially detrimental effects, remain unknown. Methods. Forty patients were prospectively randomized into either a minimally invasive (minimal) or traditional (open) saphenous vein harvest group. Smooth muscle contractile function was assessed by responses to potassium chloride and phenylephrine. Endothelial cell function was assessed by responses to serial escalations in concentration of acetylcholine, bradykinin, calcium ionophore, sodium nitroprusside, and N-nitro-L-arginine using isometric tension studies. Results. Harvest times were similar for both groups. The total incision length in the minimal group was significantly shorter than in the open group. There were no differences in smooth muscle contractions to either receptor-independent or receptor-mediated agonists between the two groups. Similarly, vasorelaxation in response to both endothelium-dependent and endothelium-independent agonists were similar in both groups. Conclusions. Minimally invasive saphenous vein harvesting is associated with similar medial smooth muscle and endothelial function as open harvesting. These findings suggest that minimally invasive harvesting techniques can be used without major detrimental effects on vascular integrity. (Ann Thorac Surg 2001;71:1503 7) 2001 by The Society of Thoracic Surgeons Coronary artery bypass grafting remains widely used for the treatment of coronary artery disease [1]. Although arterial grafts are being used with increasing frequency, the long saphenous vein remains the most frequently placed conduit. However, the long incision required for open (traditional) harvest of the saphenous vein is associated with major morbidity that can dominate postoperative recovery [2]. Impaired leg wound healing occurs in as many as 1% to 25% of patients [3]. Wound infections, hematomas, recurrent cellulitis, and saphenous neuropathy can prolong recovery [4], and the requirement of wound dressing and the difficulty in mobilization can impair a patient s quality of life. The use of minimally invasive saphenous vein harvesting has been advocated in an effort to minimize woundrelated problems [5 7]. Some evidence suggests that these techniques may reduce leg wound complications such as pain and infection [7]. Several techniques are available for minimally invasive vein harvesting, but all require traction on the vein to maximize surgical visibility and enable side branch ligation. Although endothelial cell coverage appears grossly intact after such harvesting Accepted for publication Jan 20, 2001. Address reprint requests to Mr Pillai, Oxford Heart Centre, John Radcliffe Hospital, Oxford OX3 9DU, United Kingdom. techniques [8, 9], the impact on endothelial and medial function in clinical situations is unclear. Excessive surgical manipulation of saphenous vein conduits clearly impairs endothelial cell function and reduces the bioavailability of nitric oxide (NO) [10 12]. Endothelial injury allows platelet and leukocyte adhesion that can result in graft thrombosis [13]. Furthermore, surgical preparation stimulates smooth muscle cell proliferation that exacerbates the intimal hyperplastic characteristic of accelerated atherosclerosis in diseased vein grafts [14, 15]. Thus, functional integrity of harvested saphenous vein has important implications for immediate and long-term graft survival. We therefore sought to evaluate endothelial cell function and medial smooth muscle function in saphenous vein harvested by a minimally invasive technique compared with vein harvested by the open technique. Material and Methods Forty patients admitted for elective coronary artery bypass grafting requiring long saphenous vein harvest were prospectively randomized into two groups: saphenous vein harvest by a minimally invasive technique using the SaphLITE System (Genzyme Surgical Products, Cam- 2001 by The Society of Thoracic Surgeons 0003-4975/01/$20.00 Published by Elsevier Science Inc PII S0003-4975(01)02463-8

1504 BLACK ET AL Ann Thorac Surg MINIMALLY INVASIVE VEIN HARVESTING 2001;71:1503 7 bridge, MA) (minimal group) or traditional open harvesting (open group). Ethical approval was granted by the local research ethics committee, and all patients gave informed consent for surplus vein to be kept for vasomotor studies. Clinical variables known to be important in vascular function were recorded. In addition to randomization to harvest technique group, randomization was used to determine whether harvest should begin in the calf or the groin. Harvest Techniques A single surgeon (E.A.B.) carried out all operations. The minimally invasive SaphLITE System employs a modified bridging technique. The vein was identified through a longitudinal incision either proximal to the medial malleolus or distal to the groin. A dissection plane was established anterior and posterior to the vein by gentle clearing of subcutaneous fascia. The blade of the Saph- LITE was placed into the wound, and the vein was progressively mobilized. The Genzarm (Genzyme Surgical Products, Cambridge, MA) was used to aid retraction. Side branches were ligated with Ligaclips (Ethicon, Somerville, NJ) and then divided. After dissection was carried as far as possible, an incision was made more distal to that point, and dissection was continued further up and down the limb. All incisions were closed in layers, and a pressure dressing was applied immediately. In open harvesting, the vein was identified either just proximal to the medial malleolus or distal to the skin crease in the groin. The skin was incised along the whole length of the vein, and careful dissection was used to isolate the vein in situ, with attention given not to traumatize the vein or its branches. Side branches were ligated with 4-0 silk on the vein side and Ligaclips on the patient s side. The leg wound was closed in layers and a full-length pressure dressing, applied. Vasomotor Studies Saphenous vein segments were removed prior to distention and immediately rinsed without pressure, immersed in iced oxygenated Krebs-Henseleit buffer [16], and transported to the laboratory within 30 minutes. Vein segments were divided into three or four rings approximately 4 mm long and mounted in 25-mL organ baths (Linton Instrumentation, Diss, Norfolk, UK). The tension was recorded directly onto a computer (MP100 System; Biopac Systems Inc, CA and Acqknowledge software). Optimal resting tension was determined in baseline studies. We measured contractions to both 60 mmol/l potassium chloride, (a receptor-independent agonist) and cumulative doses (10 9 mol/l to 10 4 mol/l) of phenylephrine hydrochloride (an -1 adrenergic receptor agonist) [16]. After submaximal precontraction with phenylephrine (typically 3 10 6 mol/l), relaxations were serially determined to three endothelial-dependent agonists acetylcholine (ACh), bradykinin, and calcium ionophore A23187 and to an endothelial-independent agonist, sodium nitroprusside. Dose response curves to ACh, bradykinin, A23187, and nitroprusside were determined as Table 1. Demographic and Risk Factor Profiles for the Two Groups a Variable Minimal Group Open Group (n 18) p Value Female sex 4 (18) 5 (28) 0.07 Age (y) 64 10 63 12 0.69 NYHA class 3 1 2 1 0.87 Diabetes 8 (36) 7 (39) 0.74 Smoker 16 (73) 11 (61) 0.54 PVD 7 (32) 7 (39) 0.56 Plasma cholesterol (mmol/l) 5.2 1.5 4.7 1.9 0.41 a Data are presented as absolute numbers with percentages in parentheses or as mean values the standard deviation. See Material and Methods section for more details. NYHA New York Heart Association; PVD peripheral vascular (arterial) disease. previously described [16]. All experiments were performed in the presence of indomethacin (10 mol/l) to inhibit vascular prostaglandin synthesis. Relaxation to nitroprusside was performed after addition of n-nitro-larginine (1 mmol/l) to inhibit endogenous NO synthesis [17]. Statistical Analysis Values are expressed as the mean the standard deviation or the standard error as appropriate. A p value of less than or equal to 0.05 was considered significant (Student s t test or analysis of variance as appropriate). The sensitivity of each ring to the various agonists was expressed as the effective concentration that would produce 50% of the maximal response, or EC 50 from a logarithmic dose-response plot. In all cases, n refers to the number of patients studied. An average of three rings was used from each patient. Results Demographic and Risk Factor Profiles Demographic and atherosclerotic risk factor profiles of the patient groups are shown in Table 1. The minimal and open groups were closely matched. Surgical Outcomes Sufficient vein was obtained for coronary artery bypass grafting and for analysis in all instances. As expected, minimally invasive harvest required fewer incisions and a greatly reduced total length of incision (Table 2). This technique required a slightly longer time, but the mean length of vein obtained was longer, so the rate of vein harvest (centimeters per minute) was no different using the minimal harvest technique. In 4 patients randomized to minimally invasive harvest, additional vein had to be harvested by open technique because of abnormalities of the vein (3 patients) or prolonged harvest duration (1 patient). These abnormalities were calcification of the vein in 2 patients and an obliterated lumen in 1 patient. Repairs were counted only if a side branch avulsion

Ann Thorac Surg BLACK ET AL 2001;71:1503 7 MINIMALLY INVASIVE VEIN HARVESTING 1505 needed to be repaired with a Prolene suture (Ethicon). Surgical preference (of the primary surgeon, not the vein harvester) dictated that most branches that were clipped had the clip changed to a tie. Also, in some patients in the open group, the primary surgeon s decision was that vein should be harvested from both legs, which resulted in more than one incision in this group. Saphenous Vein Contractile Function To examine medial smooth muscle function after minimally invasive or open saphenous vein harvest, we determined contractions to potassium chloride (60 mmol/ L), a receptor-independent agonist, and to the -1 adrenergic receptor agonist phenylephrine. Vascular contractions were preserved in the two groups, and maximal contractions to both potassium chloride and phenylephrine were almost identical in veins harvested by the two techniques (Fig 1). Similarly, sensitivities to phenylephrine, determined by 50% effective concentration (EC 50 ) were not significantly different ( p 0.3). Saphenous Vein Relaxation Responses As loss of endothelial function is a highly sensitive indicator of vascular injury, we sought to evaluate relaxation responses in saphenous vein rings to a range of receptor-independent and receptor-mediated agonists. Two different NO mediated receptor-dependent agonists, ACh and bradykinin, and a receptor-independent agonist, calcium ionophore A23187, caused relaxations with similar maximal responses and sensitivities (EC 50 s) in vein rings harvested by the minimally invasive or the open technique (Table 3; Fig 2). The endothelialindependent direct NO donor nitroprusside produced the same complete relaxation in both groups, thereby demonstrating preserved responsiveness of vascular smooth muscle to exogenous NO. We used the response to the NO synthase antagonist n-nitro-l-arginine (1 mmol/l) as a bioassay of basal NO production mediated by pathways independent of agonists such as ACh or bradykinin. Addition of this antagonist to the organ bath produced increases in tension of Table 2. Surgical Outcomes for the Two Groups a Variable Minimal Open p Value Duration of CPB 75.5 23.0 75.1 20.8 0.94 (min) Harvest time (min) 42.4.9 17.2 34.8 9.0 0.07 Vein length (cm) 47.6 17.2 40.1 11.8 0.13 Rate (cm/min) 1.1 0.2 1.2 0.4 0.48 No. of repairs 2.8 1.8 0.9 1.2 0.001 Incision length (cm) 12.4 5.6 40.0 11.6 0.001 No. of incisions 4.2 2.1 1.1 0.2 0.001 Vein/incision (cm/cm) 4.0 0.9 1.0 0.1 0.001 a Data are presented as the mean the standard deviation for all 40 patients. Four patients in the minimal group crossed over to the open group during harvest. The operative details are included in both columns as appropriate. CPB cardiopulmonary bypass. Fig 1. Vasoconstriction of saphenous vein rings harvested using minimally invasive (Minimal) and traditional (Open) harvest techniques. Rings of saphenous vein from both groups were mounted in an organ bath to measure isometric tension. (A) Dose response curves for phenylephrine (PE); tension is expressed as percent of maximum tension generated from 60 mmol/l potassium chloride (KCl). (B) Maximum tension generated to 60 mmol/l KCl and 10 4 mol/l PE. 27% and 33% ( p not significant) in the minimal group and the open group, respectively (Fig 3). Segments of vein from the 4 patients who crossed over were available for matched analysis of vasomotor function. There were no significant differences between the vasomotor response of this diseased vein with either harvest technique. However, because the responses were minimal in these diseased vein rings, it was considered impossible to draw inferences about the effect of harvestinduced trauma from these rings. Comment Leg wound morbidity associated with the open technique of saphenous vein harvesting has stimulated the

1506 BLACK ET AL Ann Thorac Surg MINIMALLY INVASIVE VEIN HARVESTING 2001;71:1503 7 Table 3. Maximal Relaxation of Saphenous Vein Rings and Concentration Required to Produce 50% of Maximal Response in Both Groups a Agonist Maximum Response b (%) EC 50 c ( Log [Agonist]) Minimal Open p Value Minimal Open p Value Acetylcholine 37.6 5.1 33.2 2.4 0.32 5.22 10.66 2.01 4.11 0.32 Bradykinin 65.5 7.3 54.4 3.7 0.33 5.15 10.16 4.87 9.86 0.52 A23187 66.5 8.4 54.9 8.7 0.49 2.49 4.70 2.94 5.70 0.50 Sodium nitroprusside 114.9 5.9 113.8 5.0 0.91 8.03 16.5 5.43 10.34 0.39 a Saphenous vein rings were precontracted with 10 6 mol/l phenylephrine in an organ bath set up to measure isometric tension. Dose responses to the endothelial-dependent agonists acetylcholine, bradykinin, and calcium ionophore A23187 and the endothelial-independent agonist sodium nitroprusside were then recorded. Data are shown as the mean the standard error of the mean. b Maximal relaxation is expressed as percent reduction in precontracted tension. c Note units for EC 50 are log (agonist). EC 50 effective concentration required to produce 50% of maximal effect. development of minimally invasive harvesting techniques [5, 6, 18, 19]. All minimally invasive techniques involve increased traction and the potential for vascular trauma, thus raising the concern that they may result in venous conduits with impaired vascular function. However, the principal findings of our study show that saphenous vein harvested by a minimally invasive technique has equally well preserved endothelial cell function and smooth muscle cell function as veins harvested by a careful open dissection technique, and suggest that concerns over surgical trauma may be exaggerated. Preservation of saphenous vein function during minimally invasive harvest has important implications for the immediate functional integrity of venous conduits and for the long-term patency of venous bypass grafts. Surgical preparation of saphenous vein injures the endothelial and medial layers [10 12, 14, 15, 20], reduces NO bioavailability [10], and results in platelet and leukocyte adhesion and smooth muscle cell migration into the intima [12 14]. These changes contribute to the accelerated atherosclerosis, characteristic of mature vein grafts, that leads to vein graft vascular dysfunction [21 23] and graft failure. Although endothelial cell coverage appears grossly intact after minimally invasive harvesting techniques [8, 9], studies of endothelial function after minimally invasive vein harvesting have been limited. We found that ACh vasorelaxations in both groups ranged from 30% to 40%, results suggesting good preservation of endothelial cell function in both groups. Fig 2. Endothelial-dependent and endothelial-independent vasorelaxation in saphenous vein rings harvested using minimally invasive (Minimal) and traditional (Open) harvest techniques. Human saphenous vein rings were mounted in an organ bath for isometric tension measurements. Rings were initially precontracted with 10 6 mmol/l phenylephrine. After reaching a stable tension, they were relaxed with endothelial-dependent and endothelial-independent agonists: acetylcholine (ACh), bradykinin (BK), calcium ionophore (A23187), and sodium nitroprusside (SNP). Percent of this precontracted tension is plotted against dose of agonist. Fig 3. Increase in ring tension after addition of n-nitro-l-arginine. Saphenous vein rings were mounted in an organ bath as described. After submaximal precontraction with 3 10 6 mol/l phenylephrine, 1 mmol/l n-nitro-l-arginine was added to the organ bath chambers. Increases in tension occurred as basal nitric oxide production stopped. Tension is expressed as percent of precontraction tension. Tension in the minimal group increased to 127% of precontracted tension and tension in the traditional group, to 133%.

Ann Thorac Surg BLACK ET AL 2001;71:1503 7 MINIMALLY INVASIVE VEIN HARVESTING 1507 We studied relaxations to a range of agonists. Reliance on ACh relaxations alone may affect the precision of the results. We know that certain clinical details affect the production of NO [24]. Therefore, we were interested to find that plasma cholesterol, diabetes, and other risk factors for impaired vascular function were not significantly different between the groups (see Table 1). We also compared the response of the vessels to endothelium-independent agonists to check the response in more detail. By using both ACh and bradykinin, we analyzed responses mediated by distinct receptor/g protein mediated pathways. Furthermore, endothelial NO synthase activity in response to isometric contraction, assayed by the increase in tension in precontracted rings in response to the NOs synthase inhibitor n-nitro-larginine, was used to determine the activation of endothelial NO synthase by pathways that mediate the response to shear stress in vivo [17]. Finally, we used the receptor-independent agonist calcium ionophore A23187 to maximally stimulate endothelial NO synthase independent of any receptor-mediated pathway. In all of these studies, there was no difference in the bioavailability of NO between saphenous veins harvested by a minimally invasive technique versus an open technique, thus conclusively demonstrating preservation of NO mediated endothelial cell function across a broad range of cellular signaling pathways. Assessment of medial smooth muscle cell contractile function is also important, as traction on the vein could cause overstretching of the medial layer and disrupt the contractile elements. In our study, there was no difference in contractile response between saphenous veins harvested by either the minimally invasive or open technique. In conclusion, minimally invasive saphenous vein harvesting using the SaphLITE System preserves both endothelial and medial smooth muscle function as well as open harvest does. Vein conduits harvested by minimally invasive techniques should be no more prone to vascular dysfunction or failure than those collected by open technique. We are grateful to the Oxford Regional Health Authority and Genzyme Corporation for educational grants to support this research. Drs Channon and West are supported by the British Heart Foundation. References 1. Eagle KA, Guyton RA, Davidoff R, et al. ACC/AHA guidelines for coronary artery bypass graft surgery: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines (Committee to Revise the 1991 Guidelines for Coronary Artery Bypass Graft Surgery). American College of Cardiology/American Heart Association. J Am Coll Cardiol 1999;34:1262 347. 2. Horvath KD, Gray D, Benton L, Hill J, Swanstrom LL. Operative outcomes of minimally invasive saphenous vein harvest. Am J Surg 1998;175:391 5. 3. DeLaria GA, Hunter JA, Goldin MD, Serry C, Javid H, Najafi H. Leg wound complications associated with coronary revascularization. J Thorac Cardiovasc Surg 1981;81:403 7. 4. Lavee J, Schneiderman J, Yorav S, Shewach-Millet M, Adar R. Complications of saphenous vein harvesting following coronary artery bypass surgery. J Cardiovasc Surg (Torino) 1989;30:989 91. 5. Allen KB, Shaar CJ. Endoscopic saphenous vein harvesting. Ann Thorac Surg 1997;64:265 6. 6. Cable DG, Dearani JA. Endoscopic saphenous vein harvesting: minimally invasive video-assisted saphenectomy. Ann Thorac Surg 1997;64:1183 5. 7. Davis Z, Jacobs HK, Zhang M, Thomas C, Castellanos Y. Endoscopic vein harvest for coronary artery bypass grafting: technique and outcomes. J Thorac Cardiovasc Surg 1998;116: 228 35. 8. Allen KB, Griffith GL, Heimansohn DA, et al. Endoscopic versus traditional saphenous vein harvesting: a prospective, randomized trial. Ann Thorac Surg 1998;66:26 32. 9. Cable DG, Dearani JA, Pfeifer EA, Daly RC, Schaff HV. Minimally invasive saphenous vein harvesting: endothelial integrity and early clinical results. Ann Thorac Surg 1998;66: 139 43. 10. Angelini GD, Christie MI, Bryan AJ, Lewis MJ. Surgical preparation impairs release of endothelium-derived relaxing factor from human saphenous vein. Ann Thorac Surg 1989;48:417 20. 11. Bush HL Jr, Jakubowski JA, Curl GR, Deykin D, Nabseth DC. The natural history of endothelial structure and function in arterialized vein grafts. J Vasc Surg 1986;3:204 15. 12. Lawrie GM, Weilbacher DE, Henry PD. Endotheliumdependent relaxation in human saphenous vein grafts. Effects of preparation and clinicopathologic correlations. J Thorac Cardiovasc Surg 1990;100:612 20. 13. Angelini GD, Bryan AJ, Williams HM, Morgan R, Newby AC. Distention promotes platelet and leukocyte adhesion and reduces short-term patency in pig arteriovenous bypass grafts. J Thorac Cardiovasc Surg 1990;99:433 9. 14. Soyombo AA, Angelini GD, Newby AC. Neointima formation is promoted by surgical preparation and inhibited by cyclic nucleotides in human saphenous vein organ cultures. J Thorac Cardiovasc Surg 1995;109:2 12. 15. Dilley RJ, McGeachie JK, Prendergast FJ. A review of the histologic changes in vein-to-artery grafts, with particular reference to intimal hyperplasia. Arch Surg 1988;123:691 6. 16. Channon KM, Qian H, Neplioueva V, et al. In vivo gene transfer of nitric oxide synthase enhances vasomotor function in carotid arteries from normal and cholesterol-fed rabbits. Circulation 1998;98:1905 11. 17. Fleming I, Bauersachs J, Schafer A, Scholz D, Aldershvile J, Busse R. Isometric contraction induces the Ca 2 - independent activation of the endothelial nitric oxide synthase. Proc Natl Acad Sci USA 1999;96:1123 8. 18. Newman RV, Lammle WG. Minimally invasive vein harvest: new techniques with old tools. Ann Thorac Surg 1999;67: 571 2. 19. Goel P, Sankar NM, Rajan S, Cherian KM. Use of direct laryngoscope for better exposure in minimally invasive saphenous vein harvesting. Eur J Cardio-thorac Surg 2000; 17:182 3. 20. LoGerfo FW, Quist WC, Cantelmo NL, Haudenschild CC. Integrity of vein grafts as a function of initial intimal and medial preservation. Circulation 1983;68(Suppl II):II117 24. 21. O Neil GS, Chester AH, Schyns CJ, Tadjkarimi S, Borland JA, Yacoub MH. Effect of surgical preparation and arterialization on vasomotion of human saphenous vein. J Thorac Cardiovasc Surg 1994;107:699 706. 22. Ku DD, Caulfield JB, Kirklin JK. Endothelium-dependent responses in long-term human coronary artery bypass grafts. Circulation 1991;83:402 11. 23. Cross KS, Davies MG, el-sanadiki MN, Murray JJ, Mikat EM, Hagen PO. Long-term human vein graft contractility and morphology: a functional and histopathological study of retrieved coronary vein grafts. Br J Surg 1994;81:699 705. 24. Guzik TJ, West NE, Black E, et al. Vascular superoxide production by NAD(P)H oxidase: association with endothelial dysfunction and clinical risk factors. Circ Res 2000;86: E85 90.