Human internal mammary artery produces more prostacyclin than saphenous vein

J

THoRAc CARDIOVASC SURG

92:88-91, 1986

Human internal mammary artery produces more prostacyclin than saphenous vein 1be patency rate of internal mammary artery grafts is reported to be better than that of saphenous vein grafts in myocanlial revascularizationoperatiOM. To identifya possible biochemical explanation for this phenomenon, we studied the production of prostacylin by the internal mammary artery and saphenous vein in 11 patients. Segments of internal mammary artery and saphenous vein from each patient were incubated in Krebs-Henseleit buffer at 37° C. Mter 15 minutes, the basal production of 6keto-prostaglandin F la (prostacyclin metabolite) by the internal mammary artery was 152 ± 39 pg/mg wet weight (mean ± standard error of the mean~ whereas the saphenous vein produced only 68 ± 17 pg/mg (p < 0.001). Mter 30 minutes, the internal mammary artery produced 179 ± 42 pg/mg, whereas the saphenous vein produced 75 ± 18 pg/mg (p < 0.001). Mter the basal incubation period, the vessels were incubated with arachidonic acid (prostaglandin substrate) for 15 minutes. The internal mammary artery produced 49.4 ± 9.9 pg/mg, whereas the saphenous vein produced only 22.6 ± 9.8 pg/mg (p < 0.01). These obsenatiom suggest that the capacity of the internal mammary artery to produce prostacyclin in both a basal and a stimulated state is greater than that of the saphenous vein. Since prostacyclin is a potent vasodilator and inhibitor of platelet function, these results provide a possible biochemical explanation for the clinically observed better patency rate of internal mammary artery grafts,

Amer Chaikhouni, M.D., Fred A. Crawford, M.D., Pamela J. Kochel, M.S., Lawrence S. Olanoff, M.D., Ph.D., and Perry V. Halushka, Ph.D., M.D., Charleston. S. C.

I

n patients with coronary arterial occlusive disease, coronary bypass grafting provides good symptomatic palliation in most surgical candidates and prolongs life in some specificsubgroups.I. 2 Long-term follow-up'< has shown superior patency of internal mammary artery (IMA) bypass grafts compared to saphenous vein (SV) grafts. These studies, along with the development of techniques (bilateral IMA, sequential IMA, free graft lMA) that allow the lMA to be used for essentially all coronary artery branches, have stimulated increased interest in IMA grafts. The excellent patency of lMA grafts is largely attributed to several factors, including From the Division of Cardiothoracic Surgery and the Department of Pharmacology at the Medical University of South Carolina, Charleston, S. C. Supported in part by Grants HL29566 and RRI070. Received for publication July 16, 1985. Accepted for publication Aug. 13, 1985. Address for reprints: Dr. Fred A. Crawford, 171 Ashley Ave., Charleston, S. C. 29425.

88

preservation of lymphatics and vasa vasorum in the pedicle. However, these factors do not explain the superior patency of IMA when used as a free graft' or when its pedicle is dissected away, such as when used as a sequential graft" or during the preparation of the distal end of lMA for anastomosis. The importance of intact endothelium in the prevention of intravascular thrombosis was established by Virchow. The remarkable antithrombotic function of intact endothelium has been attributed in part to the production of prostacylin (prostaglandin 12) a potent vasodilator and inhibitor of platelet aggregation." Prostacyclin is labile and spontaneously hydrolyzes to the stable but inactive 6-keto prostaglandin F la (6-ketoPGF 1a ) · Since endothelium in different parts of the vascular tree differs in its ability to produce prostacyclin," we attempted to identify a possiblebiochemicalexplanation for the reported superior patency of lMA grafts. The hypothesis that we tested was that endothelium in the IMA produces more prostacyclin than that in SVs.

Volume 92 Number 1 July. 1986

Table I. Production of immunoreactive 6-keto-PGFl a by human saphenous vein (SY) and internal mammary artery (IMA) segments during the initial 30 minute basal period

Table D. Arachidonic acid stimulation of immunoreactive 6-keto-PGFl a by human saphenous vein (SY) and internal mammary artery (IMA) segments

Immunoreactive 6-keto-PGFlo (pg/mg wet weight)

SV (0 = 11) IMA (0 = 11)*

89

Prostacyclin

/5 min

30 min

68 ± 17 152 ± 39

75 ± 18 179 ± 42

Legend: Data are expressed as mean ± standard error of the mean. 'p < 0.001 when compared with SV (analysis of variance).

Methods Remnant segments of dilated and undilated SV and remnant segments of IMA from 11 adult male patients undergoing coronary bypass graft operation were obtained. Each of these patients was receiving multiple antianginal drugs, such as beta blockers, calciumchannel blockers,and nitroglycerin, but none was receiving nonsteroidal anti-inflammatory drugs. The SV was dilatedwith heparinized lactated Ringer's solution using low pressure at room temperature. The IMA was not dilated mechanically, but it was covered with papaverine-soaked gauze in preparation for grafting. The remnant segments were transported to the laboratory in lactated Ringer's solution. After removal of the adventitia, the vessels were cut into four lengths, 0.5 em each, and opened longitudinally to expose the endothelium. Eachpiece of tissue was placed into 4.0 .ml of incubation medium in a 25 ml Erlenmeyer flask and incubated with constantshaking for 30 minutes at 37 C. This incubation period established the basal rates of prostacyclin production and allowed for stabilization of the synthesis rate. The incubation medium consisted of a KrebsHenseleit bicarbonate buffer (NaCl, 118 mmoljL; KCl, 5.4 mmoljL; MgS04, 1.0 mmoljL; CaCI 2, 2.5 mmoljL; Na2HP04, 1.1 mmoljL; NaHC03, 25 mmol/L; dglucose, 10 mmoljL) and was bubbled with oxygen and carbondioxide (95:5%). During the stabilization period, aliquots ofthe media were taken, 0.5 rnl each, at 15 and 30 minutes and frozen at -20 C until assay. After the 30 minute basal period, the tissue was transferred to a second flask containing either arachidonic acid (10 ~mol/L) or vehicle. The arachidonic acid stock solution (100 mmoljL) was prepared fresh in O.1N NaOH : ethanol (1:1, volume/volume) and was diluted to 1 mmol/L in O.1N NaH 2P04 buffer, pH 8.0. Aliquots, 0.5 rnl each, were removed from the bathing media at 5, 10, and 15 minutes and frozen at -20 0 C until assay. The vessels were removed from the flask, blotted dry, 0

0

Immunoreactive 6-keto-PGFl o (pg/mg wet weight) 15 min

±AA SV (0

= 8)

IMA*t (0

= 8)

+ +

5.7 10.7 8.9 21.8

± ± ± ±

1.5 4.5 2.4 6.6

7.2 18.3 16.2 34.4

± ± ± ±

2.3 7.7 3.9 7.9

10.5 22.6 23.3 49.4

± ± ± ±

3.2 9.8 5.4 9.9

Legend: Data are expressed as mean ± standard error of the mean. AA, Arachidonic acid. 'p < 0.01 for the IMA compared to SV in the presence of arachidonic acid (analysis of variance). tp = 0.001 for the IMA compared to SV in the absence of arachidonic acid (analysis of variance).

and weighed. Immunoreactive 6-keto-PGF t a (a stable hydrolysis product of prostacyclin) was determined by a previously described radioimmunoassay procedure." The results were reported as picograms of immunoreactive 6-keto PGF t a per milligram of wet weight. The study protocol was approved by the institutional review beard of the Medical University of South Carolina.

Statistical analysis Log transformations of the synthesis data were performed before a two-way analysis of variance. Because each vessel segment was divided into four lengths, each experimental value within the basal period represented the mean of quadruplicate determinations. Each experimental value within the arachidonic acid-stimulated period represented the mean of duplicate determinations.

Results When lengths of SV and IMA were initially incubated in Krebs-Henseleit buffer for 30 minutes, immunoreactive 6-keto-PGF t a appeared spontaneously in the media (Table I). The IMA synthesized significantly greater amounts (p < 0.001) of immunoreactive 6keto-PGF t a than the SV at both 15 and 30 minute time points. Control (unstimulated) production of immunoreactive 6-keto-PGF ta by the vessels during the second 15 minutes was lower than that seen during the initial equilibration period. However, there was an increased accumulation of immunoreactive 6-keto-PGF t a in the media with time (p < 0.05) (Table II). Arachidonic acid, the precurser for prostacyclin, stimulated immunoreactive 6-keto-PGF ta synthesis significantly more than

9 0 Chaikhouni et al.

the vehicle treatment in both SV and IMA. When the vessels were compared directly, during either the vehicle or arachidonic acid exposure, the IMA produced significantly more immunoreactive 6-keto-PGF la than the SV (Table 11). The possibility existed that dilating the SV altered its synthesis of prostacyclin. Therefore, the synthesis of prostacyclin by dilated and undilated SVs was compared in veins taken from four patients. Basal production of immunoreactive 6-keto-PGF l a by undilated SVs (n = 4) was 250 ± 77 and 278 ± 91 pg/rng for 15 and 30 minute time points, respectively. Production of immunoreactive 6-keto-PGF la by dilated SVs (n = 4) obtained from the same patients was 144 ± 53 and 171 ± 67 pg/rng for the 15 and 30 minute time points, respectively. In the presence of arachidonic acid, the undilated SVs produced 43.1 ± 22.1, 66.3 ± 34.1, and 98.8 ± 46.3 pg/rng of immunoreactive 6-keto-PGF la at the 5, 10, and 15 minute time intervals, respectively. By contrast, the dilated SVs produced 43.6 ± 21.3, 73.4 ± 30.2, and 97.3 ± 39.2 pg/rng of immunoreactive 6keto-PGF l a for the 5, 10, and 15 minute time intervals, respectively (n = 3 experiments). These differences between dilated and undilated SV segments were not statistically significant. Discussion

Although early results of SV coronary bypass grafting have been good, long-term follow-up has demonstrated a 47.2% closure rate of SV grafts in 10 years.' Failure of SV grafts is attributed to intimal hyperplasia and accelerated atherosclerosis, which may be demonstrated as early as 1 year postoperatively.I I Endothelial trauma produced by ischemia, by vein preparation techniques, or by arterialization hemodynamics can stimulate platelet aggregration and fibrin deposition. Platelets may release a mitogenic factor that promotes the proliferation of vascular smooth muscle cells, intimal hyperplasia, and accelerated atherosclerosis.I I Patency of the IMA grafts was better than that of the SV grafts." This superior patency of IMA grafts was demonstrated whether they were used as bilateral pedicles,' sequential grafts," or free grafts.' Several theories have been proposed to explain the difference in patency of IMA and SV grafts. The preservation of vasa vasorum and lymphatic drainage in the intact IMA pedicle is a commonly reported theory, but it does not explain the excellent patency of free IMA grafts. Another possible explanation is the similarity in size between the IMA and coronary arteries. However, free radial artery grafts to the coronary arteries failed despite good match of the luminal diameters. The elimination of

The Journal of Thoracic and Cardiovascular Surgery

the proximal anastomosis is a possible explanation. However, free IMA grafts also have good patency despite having a proximal anastomosis. Since mechanical factors such as these cannot explain the superiority of the IMA graft, we postulated that this remarkable ability of the IMA to remain patent may be related to the ability of its endothelial lining to produce more prostacyclin. Virchow, Osler, and many others believed that intact endothelium is an important factor in the prevention of thrombosis. The remarkable antithrombotic ability of endothelium is attributed in part to the ability of the endothelial cells to produce prostacyclin, which is a potent vasodilator and inhibitor of platelet adherence and aggregation. Various areas of the vascular system are different in their ability to produce prostacyclin,? with arteries, in general, being able to produce more prostacyclin than veins." In this study, the IMA produced more 6-keto-PGF la than the dilated SV in each of the 11 patients, both in the basal state and after incubation with arachidonic acid. We compared the IMA to the dilated rather than undilated SV to evaluate prostacyclin production by these vessels, because that is how they are used clinically. We observed that the production of prostacyclin by undilated SV segments was the same as that by dilated SV segments of the same patients in the basal and arachidonic acid-stimulated state. Mehta and Roberts" studied prostacyclin and thromboxane A, production by segments of IMA and SV in five patients. They observed that the IMA produced significantly less prostacyclin in the basal state and that the IMA produced more thromboxane A z than the SV when incubated with arachidonic acid. The reasons for the marked differences between our results and those reported by Mehta and Roberts are uncertain. This difference may be related to the preparation and measurement techniques or may be due to wide variation in prostacyclin production by these vessels, coupled with a small sample size in their study. It is not stated whether they obtained both IMA and SV segments from the same patients, as we did, or from different patients. It is also not clear whether the SV segments were obtained from dilated or undilated veins. We observed wide variations in prostacyclin production by these vessels, especially IMA, from different patients. Whether the wide array of drugs the patients were receiving influenced the synthesis rates is unknown. However, in each of our 11 patients, IMA produced more prostacyclin than its corresponding SV, both in the basal and in the stimulated states. We believe that the results reported here are in accordance with the gener-

Volume 92 Number 1 July, 1986

ally accepted finding that arteries produce more prostacyclin than veins" and offer a possible explanation for the widely reported clinical results documenting the superior patency of IMA grafts." Clearly, these observations merit further investigation. The superior patency of IMA grafts to SV grafts in coronary bypass operations is now well documented. This phenomenon has been attributed to various anatomic and surgical factors. We have demonstrated that IMA produces more prostacyclin than SV, and this observation suggests a possible biochemical explanation of the clinically observed findings and attributes the better patency of the IMA grafts to the conduit itself, whether it is used as a pedicle or as a free graft. The statistical analysis was done by C. Boyd Loadholt, Ph.D., Professor of Biometry at the Medical University of South Carolina. REFERENCES Loop FD, Cosgrove DM, Lytle BW, Thurer RL, Simpfendorfer C, Taylor PC, Proudfit WL: An 11 year evolution of coronary arterial surgery (1967-1978). Ann Surg 190:444-455, 1979 2 Rahimatoola SH: Coronary bypass surgery for chronic angina-1981. Circulation 65:225-241, 1982 3 Lytle BW, Loop FD, Cosgrove DM, Ratliff NB, Easley K, Taylor PC: Long-term (5 to 12 years) serial studies of internal mammary artery and saphenous vein coronary bypass grafts. J THORAC CARDIOVASC SURG 89:248-258, 1985 4 Jones JW, Ochsner JL, Mills NL, Hughes L: The internal mammary bypass graft. A superior second coronary artery. J THORAC CARDIOVASC SURG 75:625-631, 1978

Prostacyclin 9 1

5 Grondin CM, Campeau L, Lesperance J, Enjalbert M, Bourassa MG: Comparison of late changes in internal mammary artery and saphenous vein grafts in two consecutive series of patients 10 years after operation. Circulation 70:Suppl 1:208-212, 1984 6 Singh RN, Sosa JA, Green GE: Long-term fate of the internal mammary artery and saphenous vein grafts. J THoRAc CARDIOVASC SURG 86:359-363, 1983 7 Lytle BW, Cosgrove DM, Saltus GL, Taylor PC, Loop FD: Multivessel coronary revascularization without saphenous vein. Long-term results of bilateral internal mammary artery grafting. Ann Thorac Surg 36:540-548, 1983 8 Tector AJ, Schmahl TM: Techniques for multiple internal mammary artery bypass grafts. Ann Thorac Surg 38:281286, 1984 9 Moncada S, Vane JR: Prostacyclin in the cardiovascular system. Adv Prostaglandin Thromboxane Res 6:43-60, 1980 10 Wise WE, Cook JA, Eller J, Halushka PV: Ibuprofen improves survival from endotoxic shock in the rat. J Pharmacol Exp Ther 215:160-164, 1980 11 Grondin CM, Lesperance J, Bourassa MG, Pasternac A, Campeau L, Grondin P: Serial angiographic evaluation in 60 consecutive patients with aorto-coronary artery vein grafts 2 weeks, 1 year, and 3 years after operation. J THoRAc CARDIOVASC SURG 67:1-6, 1974 12 Kamath ML, Matysik LS, Schmidt DH, Smith LL: Sequential internal mammary artery grafts. Expanded utilization of an ideal conduit. J THoRAc CARDIOVASC SURG 89:163-169, 1985 13 Skidgel RA, Printz MP: PGI 2 production by rat blood vessels. Diminished prostacyclin formation in veins compared to arteries. Prostaglandins 16:1-15, 1978 14 Mehta J, Roberts A: Human vascular tissues produce thromboxane as well as prostacylcin. Am J Physiol 244:R839-R844, 1983