Prostacyclin Production by Different Human Grafts Employed in Coronary Operations Andrea Sala, MD, Paolo Rona, MD, Giulio Pompilio, MD, Alessandro Parolari, MD, Carlo Antona, MD, Paolo Biglioli, MD, Giuseppe Rossoni, PhD, Laura M. Villa, PhD, and Ferruccio Berti, PhD Department of Cardiac Surgery, Centro Cardiologico, I. Monzino Fundation, and Department of Pharmacology, Chemotherapy and Medical Toxicology, University of Milan, Milan, Italy

Segments of human saphenous vein, internal mammary artery, right gastroepiploic artery, and inferior epigastric artery were incubated in vitro in Krebs-Henseleit solu- tion and compared in terms of their capacity to generate and release into the medium 6-keto-prostaglandin F,, (PGF,,), the stable metabolite of prostacyclin. The four vascular conduits were also challenged with endothe- lin-l(40 ng/mL), and accumulation of the lipidic material in the bathing fluid was also studied. The results ob- tained show clearly that under both normal and endothe- lin-1-stimulated conditions, the four vascular segments generate a substantial amount of 6-keto-PGF1,. Multiple- comparisons analysis of the results indicates that the rank order in producing 6-keto-PGF1, is as follows: inferior epigastric artery > internal mammary artery > right gastroepiploic artery > saphenous vein ( p < 0.01).

he search for a suitable vascular conduit that can yield T long-term patency in myocardial revascularization has become more and more pressing. The saphenous vein (SV) has been widely used as a bypass graft, but long- term follow-up studies [l-31 have shown a superior pa- tency rate for internal mammary artery (IMA) grafts compared with SV grafts. Consequently, the IMA has become the material of first choice for revascularization, especially when grafted on the left anterior descending coronary artery. The current tendency in coronary bypass procedures is to try to accomplish complete myocardial revascularization for severe multivessel disease by means of expanded use of IMA grafts and other arterial conduits. Moreover, some reports have described the biochemical basis for the reported superior patency rate of the IMA, that is, the IMA endothelium produces more pros- taglandin I,, or prostacyclin (PGI,) than does the SV [4].

Also the right gastroepiploic artery (GEA), which has been used for myocardial implantation [5] and as an in situ graft for direct coronary anastomoses [a], i s now considered a very promising conduit. Recently, Oku and associates [9] suggested that patency of the GEA might be greater than that of the SV, as the former shows a better

Accepted for publication Aug 10, 1993.

Address reprint requests to Dr Sala, Department of Cardiac Surgery, Centro Cardiologico, I. Monzino Fundation, Via Parea 4, 20138 Milan, Italy.

A similar order of potency was obtained in vascular conduits stimulated with endothelin-1. The rate of for- mation of immunoreactive 6-keto-PGF,, under both nor- mal and stimulated conditions by the inferior epigastric artery (normal, 301 f 8 pg/mg of tissue; stimulated, 519 f 15 pg/mg of tissue) was at 10 minutes more than 2 times (p < 0.01) that of the saphenous vein and about 1.5 times ( p < 0.01) that of the right gastroepiploic artery. In conclusion, the fact that the endothelium of the inferior epigastric artery has a better capacity to generate prosta- cyclin compared with the other vascular segments con- sidered may have some relevance with respect to its resistance to atherosclerosis and its patency rate and may indicate this arterial conduit is a good alternative graft in myocardial revascularization.

(Ann Thorac Surg 1994;57:1147-50)

capacity to generate PGI,. Prostacyclin is indeed a potent vasodilator and markedly inhibits platelet aggregation. Therefore the tonic release of this lipidic substance from intact vascular endothelium is a critical point in the prevention of intravascular thrombosis [ 101.

Comparative studies [ll] of various conduits for coro- nary revascularization have pointed out that the human inferior epigastric artery (IEA) should also be considered. According to the histologic findings, the IEA as well as the GEA may provide superior patency compared with the radial artery, for instance, which has a thicker media and is more prone to ischemia [ll]. However, because no data are available on the PG1,-releasing capacity of the IEA and because the endothelium of different parts of the vascular tree differs in its ability to form this lipidic substance, the hypothesis that the endothelium of the IEA may generate more PGI, than the SV and other arterial conduits, such as the GEA and IMA, was examined.

Material and Methods A total of 20 segments of human SV, IMA, GEA, and IEA were harvested from 20 select male patients (aged 45 to 65 years) undergoing coronary revascularization who had not received aspirin or other related compounds for 1 week. These segments were immediately frozen in liquid nitrogen and kept at -70°C until assayed. This freezing procedure, in line with that described by Oku and col-

0 1994 by The Society of Thoracic Surgeons 0003-4975/94/$7.00

1148 SALAETAL PROSTACYCLIN PRODUCTION BY CORONARY GRAFTS

Ann Thorac Siirg 1994;571147.-50

leagues [9], did not affect viability of the tissue. The vascular segments, soaked in Krebs-Henseleit bicarbonate buffer, were cut in small pieces (0.5 to 1.0 cm in length), weighed, and incubated singly in 3 mL of Krebs-Henseleit solution of the following composition (millimoles per liter): NaCl, 118; KCl, 5.4; MgS04, 1; CaCl,, 2.5; Na,HP04, 1.1; NaHCO,, 25; and glucose, 10. This solu- tion was bubbled with a mixture of 5% carbon dioxide and 95% oxygen, and the final pH was adjusted to 7.4.

After 30 minutes of equilibration, the bathing fluid was changed, and aliquots of 0.5 mL were drawn at 0, 2.5, 5, 10, and 20 minutes. These aliquots were put into 20 mL of 15% C,H50H with 200 pL of 1 N HCl. This medium was then passed through a Sep-Pak C,, cartridge, and 6-keto- prostaglandin F,, (PGF,,) was selected using reversed- phase high-performance liquid chromatography and dried. 6-keto-PGF1,, the stable hydrolysis product of PGI,, was measured by enzyme immunoassay [12]. In particular, 6-keto-PGF1, kits (catalog no. 416011; Cayman Chemical Company, Ann Arbor, MI) were utilized. The total amount of 6-keto-PGF1, released by the vascular segments into the bathing fluid for each incubation period was expressed in picograms per milligram of dried tissue.

Following the same protocol, a number of experiments were performed in the presence of 40 ng/mL of endothe- lin-1 (ET-I), added at time 0, to establish the responsive- ness of the four vascular segments under examination to the well-known PG1,-releasing ability of this polypeptide

The data for the generation of 6-keto-PGF1, by the four vascular conduits under both normal and ET-1-stimulated conditions were processed according to Duncan's test [ 141 for multiple-comparisons analysis. This test also was used for the evaluation of the time-dependent release of 6-keto- PGF,, in each vascular segment.

1131.

Results When lengths of human IEA, IMA, GEA, and SV were incubated in Krebs-Henseleit buffer for 20 minutes, im- munoreactive 6-keto-PGF1, appeared in the medium. The phenomenon was time dependent and markedly potenti- ated by ET-1 (40 ng/mL) (Figs 1, 2). The kinetic profile of the accumulation of the lipidic material attained its peak at 10 minutes. For each vascular conduit, the difference between normal (unstimulated) and ET-1-stimulated con- ditions was significant ( p < 0.01) (Fig 3).

As shown in Table 1, the IEA displayed a greater capacity to produce 6-keto-PGF1, compared with the other vascular segments examined. In fact, at 10 minutes, under both normal and stimulated conditions, the IEA was ranked first and was followed in rank order by the IMA, GEA, and SV ( p < 0.01). The results obtained from multiple-comparisons analysis of area-under-the-curve data, in both the absence and the presence of ET-1, indicated a significant difference ( p < 0.01), the rank order among the vascular segments being as follows: IEA > IMA > GEA > SV. Note that the rate of formation of

T

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0 2.5 5 10

T i m e ( min Fig I . Kinetic profile of the spontaneous release

IEA

IlHA

GEA

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I

20

f 6-kto-pros- ), internal mam- taglandin F,, (PGF,,) by inferior epigastric (IE

m a y artery (IMA), right gastroepiploic artery (GEA), and saphenous vein (SV) incubated in vitro. Each point represents the mean ? the standard error of the mean of six different vascular segments. Multi- ple-comparisons analysis carried out for each vascular segment acc80rd- ing to the Duncan test show a significant time-dependent increase in 6-keto-PGF1 a production.

immunoreactive 6-keto-PGF1, at 10 minutes by the IEA, under both normal and stimulated conditions, was more than 2 times that of the SV (p < 0.01) and about 1.5 times that of the GEA ( p < 0.01) (see Table 1).

- 6oo 1 W a M y1 .- - I

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. E" 400{ er

GEA

sv

0 2.5 5 10 20

T i m e ( min 1 Fig 2. Kinetic profile of the release of 6-kto-prostaglandin Flu (PGF,,) by inferior epigastric artery (IEA), internal mammary artery (IMA), right gastroepiploic artery (GEA), and saphenous vein (SV) stimulated in vitro by endothelin-2 (ET-I). Each point represents the mean t the standard error of the mean of six difierent vascular seg- ments. The ET-I was added to the incubation medium at the final concentration of 40 nglmL. Multiple-comparisons analysis carried out for each vascular segment according to the Duncan test show a :jignifi- cant time-dependent increase in 6-kto-PGFIa production.

Ann Thorac Surg 1994;571147-50

SALA ET AL 1149 PROSTACYCLIN PRODUCTION BY CORONARY GRAFTS

ous among the various vascular segments, has a great relevance to the maintenance of the patency of blood vessels and the fluidity of blood. In fact, PGI,, the major member of the prostaglandin family formed by endothe- lial cells [15], has antiplatelet and vasodilator activity and plays a role in the regulation of leukocyte accumulation in the vessel walls as well as in the control of smooth muscle

CD

0

s v GEA IMA I E A Fig 3. Total amount (area under the curve) of 6-keto-prostaglandin F,, (PGF,,) released in 20 minutes by segments of inferior epigastric artery (IEA), internal mammary artery (IMA), right gastroepiploic artery (GEA), and saphenous vein (SV) incubated in vitro under nor- mal (B) and stimulated (endothelin-1, 40 nglmW (El) conditions. Columns represent the area under the curve (mean value * the stan- dard error of the mean) of six different vascular segments. The area under the curve was evaluated by trapezoid method (data from Figs 1, 2). Significant differences (p < 0.01) between normal and stimulated conditions for each vascular segment were found (unpaired Student t test).

Comment The results obtained from these experiments clearly indi- cate that segments of human IEA, IMA, GEA, and SV, when properly incubated in vitro, release in a time- dependent manner a substantial amount of immunoreac- tive 6-keto-PGF1,, the stable metabolite of PGI,. This biochemical event, which may be more or less conspicu-

Table 1. Generation of 6-keto-Prostaglandin F,, by Segments of Inferior Epigastric Artery, Internal Mamma ry Ar te ry, Right Gastroepiploic Artery, and Saphenous Vein Incubated In Vitro Under Normal and Stimulated Conditionsad

Peak AUC , Vascular Segment Normal Stimulated Normal Stimulated

IEA 301.2 5 8.0 519.4 2 14.5 5,120 -t 97 8,623 2 125 IMA 251.7 f 6.3 418.8 f 9.0 4,296 f 74 6,801 f 94 GEA 200.0 ? 8.6 318.9 ? 9.3 3,318 2 121 4,905 2 41 sv 130.2 2 5.0 245.1 2 12.2 2,116 2 66 3,719 2 128

proliferation and cholesterol metabolism [ 161. Loss of PGI, biosynthesis by the vasculature may be a crucial biochemical step in the development of atherosclerosis and thickening of the vascular wall [17].

The present findings also indicate that the.IEA has a better capacity to accumulate 6-keto-PGF,, in the medium than the other vascular conduits under investigation. This finding suggests that under in vivo conditions, chemical or mechanical perturbation (such as pulsatile pressure and endogenous mediators) of the endothelial cell mem- branes of this vessel may lead to a more advantageous activation of the eicosanoid system with preferential gen- eration of PGI,. In this respect, it has been demonstrated that the contraction induced by ET-1 (20 ng/mL) in spirals of human SV was almost doubled in preparations where cyclooxygenase activity and PGI, generation were im- paired by indomethacin [13].

Another point of interest arising from the present results is that the spontaneous generation of 6-keto-PGF,, by the IEA is greatly enhanced by ET-1. This observation represents an indirect indication in favor of the IEA, which seems to possess a more competent modulatory mechanism against vasoconstrictive stimuli than the other vascular conduits.

The mechanism involved in the activation of the ei- cosanoid system observed with ET-1 in the IEA is far from clear. A possible explanation is that the CaZf accumula- tion in vascular endothelial cells caused by ET-1 could enhance arachidonic acid availability from increased phospholipid deacylation [18]. In this regard, Ca2+ mobi- lization from intracellular binding sites has been proposed as a mode of action of thrombin on PGI, synthesis in vascular tissue [19].

The SV, IMA, and GEA are the conduits used most often for coronary bypass grafting. Recently, however, short-term angiographic results have demonstrated that the IEA is an acceptable free graft for myocardial revas- cularization [20-221. The fact that the IEA is very similar to the IMA in terms of its resistance to atherosclerosis [23] and the fact that the IEA has a remarkable capacity to generate PGI, (present findings) strengthen the idea that this arterial conduit should be considered a valid alterna- tive graft in patients with coronary artery occlusive dis- ease.

a Data are shown as the mean ? the standard error of the mean of six different vascular segments. Stimulated condition was established with 40 ng/mL of endothelin-I. Multiple-comparison results carried out with the Peak and AUC data according to the Duncan test show significant differences ( p < 0.01) among vascular segments. AUC = area under the curve (evaluated by trapezoid method: ordinate, 6-keto-prostaglandin F,, in 20-minute incubation periods; abscissa, time in minutes from 0 to 20); IEA =inferior epigastric artery; IMA = internal mammary artery; Peak = release of 6-keto-prostaglandin F,, in picograms per milligram of tissue at 10 minutes; SV = saphenous vein.

GEA = gastroepiploic artery;

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