Ann Thorac Surg 1996;61:143-148
© 1996 The Society of Thoracic Surgeons
Original Articles: Cardiovascular
Reduced Prostacyclin and Increased Leukotriene B4 Synthesis in Porcine Venous-Arterial Grafts
Jamie Y. Jeremy, PhD,
Mohammad Bashar Izzat, FRCS,
Sonia D. Birkett, BSc,
David M. Knight, BSc,
Alan J. Bryan, FRCS,
Gianni D. Angelini, FRCS
Bristol Heart Institute, University of Bristol, Bristol, United Kingdom
Accepted for publication September 5, 1995.
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Abstract
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Background. Migration and proliferation of vascular smooth muscle cells in the intima and superimposed atheroma are the main changes underlying late failure of saphenous vein bypass grafts. There is evidence that these events are partly modulated by complex interactions between inhibitors of vascular smooth muscle cell proliferation, such as prostacyclin (PGI2), and mitogens, such as leukotriene B4 (LTB4). Because the relative balance between these eicosanoids may play a role in vein graft failure, the synthesis of PGI2 and LTB4 was measured in porcine saphenous vein-carotid artery grafts 4 weeks after implantation and compared with ungrafted vein and common carotid artery from the same animal.
Methods. Vessels were cut into 2-mm squares and preincubated in Dulbecco's minimum essential medium for 4 hours at 37°C. Tissues were then further incubated with Dulbecco's minimum essential medium containing a range of concentrations of noradrenaline, arachidonate, and calcium ionophore A23187. Release of PGI2 and LTB4 into the supernatant was then assessed by radioimmunoassay.
Results. In response to all stimulators, PGI2 release was markedly diminished in vein grafts compared with ungrafted saphenous veins and carotid arteries. The patterns of responses were similar in each vessel type. In contrast, LTB4 release was significantly enhanced in vein grafts compared to ungrafted saphenous veins and carotid arteries.
Conclusions. These data indicate that there is a down-regulation of cyclooxygenase or PGI2 synthase in porcine vein grafts, which may constitute a further phenotypic change that would augment the hyperplastic process. Local increases in LTB4 synthesis in the vein graft, which indicates an induction of lipoxygenase and LTB4 synthase enzymes (and possibly reflects release from leukocytes which have infiltrated the graft), may contribute to increased intimal proliferation by direct promitogenic effects on smooth muscle cells.
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Introduction
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Autologous saphenous vein continues to be the most commonly used conduit for coronary artery bypass grafting [1, 2]. A significant proportion of vein grafts, however, undergo late occlusion due to migration and proliferation of vascular smooth muscle cells into the intima and superimposed atheroma [1, 2]. The mechanisms underlying these events remain to be fully clarified [3]. It is well established, however, that both mitogens (eg, platelet-derived growth factor) and vasoconstrictors (eg, noradrenaline and angiotensin II) generated by platelets, leukocytes, and vascular cells, play key roles in initiating and maintaining myointimal proliferation [4, 5].
Mitogens and vasoconstrictors also stimulate the synthesis of substances in blood vessels, which may limit or prevent smooth muscle cell proliferation. These include prostacyclin (PGI2) and prostaglandin E2 (PGE2) [5–7], the effects of which are mediated by activation of adenylyl cyclase, the synthesis of adenosine 3`5` cyclic adenosine monophosphate, and activation of specific protein kinases [8, 9].
The intracellular mechanisms governing the synthesis of PGI2 are similar to those controlling the signal transduction events that mediate mitogen and vasoconstrictor action (G protein-tyrosine kinase activation, inositol trisphosphate generation, and increase in cytosolic calcium, and protein kinase C activation) [5, 9]. It has therefore been suggested that the concomitant activation of PGI2 and other prostaglandins may constitute a negative feedback mechanism by which vascular smooth muscle cells phenotype may, at least in part, be controlled [5, 6]. Blood vessels also possess the capacity to synthesize leukotrienes (LTs), albeit much lower amounts than PGI2 [7]. Leukotrienes differ from prostaglandins in that they promote proliferation of vascular smooth muscle cells in culture at extremely low concentrations [11, 12].
Because the relative concentrations of these eicosanoids of opposing action may play a role in the pathophysiology of vein graft failure, the synthesis of PGI2 and LTB4 was assessed in porcine saphenous vein-carotid artery bypass grafts 4 weeks after implantation and compared with ungrafted saphenous veins and carotid arteries. Eicosanoid synthesis was elicited with stimulators (eg, noradrenaline, calcium ionophore A23187, and arachidonate) which act at different sites in the receptor activation-PGI2 synthesis pathway. Using this approach, it may be possible to determine whether alterations occur not only at the level of cyclooxygenase/lipoxygenase or synthases, but also at related but distal levels (surface receptors and associated signal transduction systems).
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Material and Methods
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Surgical Procedures
Studies were performed using white Landrace pigs initially weighing 25 to 30 kg in accordance with British Home Office Animal Care regulations. Premedication, anesthesia, and autologous saphenous vein into carotid artery interposition graft were performed as detailed previously [13]. Briefly, a longitudinal incision was made on the outer aspect of the hind leg to expose approximately 10 cm of the long saphenous vein. The vein was dissected free of surrounding tissue by means of a ``no touch'' technique [14] and all side branches were secured with a 6-0 Prolene ligature (Ethicon Inc, Somerville, NJ). After this, the vein was removed from the animal and irrigated with heparinized isosmotic sodium chloride (NaCl) solution (0.9 g/L). Veins were grafted by an end-to-end anastomotic technique as follows. A segment of one common carotid artery was exposed through a longitudinal neck incision (medial to sternocleidomastoid muscle) and a 3- to 4-cm segment isolated between vascular clamps was excised; both cut ends were then beveled to approximately 45 degrees. A segment of saphenous vein was similarly beveled and then anastomosed to the carotid artery with continuous 7-0 Prolene suture. The neck and leg wounds were then closed in layers and the animals allowed to recover. After 4 weeks, the pigs were reanesthetized, the neck wound reopened, and the graft identified. The carotid artery was transected distal to the graft and the absence of any blood flow was taken to indicate graft occlusion. Only patent grafts were used for further analysis. Intact carotid artery from the opposite ungrafted side was also excised, as well as the saphenous vein from the contralateral hind leg. All vessels were gently irrigated with prewarmed (37°C) Dulbecco's minimum essential medium (DMEM; Sigma Chemical Co, Poole, Dorset, UK) pregassed with 95% O2 and 5% CO2 and placed immediately in prewarmed DMEM for a period of 20 to 30 minutes before preparation of tissues for experimentation.
Preparation of Tissues and Assessment of Eicosanoid Synthesis
Vascular tissues were prepared for assessment of eicosanoid synthesis as previously described [15]. Adventitia was carefully removed from all vessels which were opened with a longitudinal incision. Each vessel was then cut laterally into approximately 2-mm strips and further into approximately 2-mm squares. Tissues were then placed in DMEM, pregassed with 95% O2, 5% CO2, and incubated at 37°C for 4 hours with frequent changes of medium to allow eicosanoid release due to trauma of preparation to subside. After preincubation, tissues in duplicate were sequestered into polypropylene tubes containing DMEM and various concentrations of drugs. Before comparison of PGI2 synthesis in ungrafted control saphenous veins, carotid arteries, and vein grafts, the effect of a range of intracellular second messenger signal activators and agonists, known to stimulate the eicosanoid in other species (including rats, rabbits, and humans) were investigated: adrenaline, noradrenaline, angiotensin I and II, histamine, serotonin, phorbol ester dibutyrate, sodium fluoride, calcium ionophore A23187, thapsigargin, and arachidonate (Sigma Chemical Co, Poole, Dorset). Tubes were incubated for a further 60 min at 37°C and supernatants removed for measurement of 6-oxo-PGF1
and LTB4 by radioimmunoassay as previously described [21]. For radioimmunoassays, polyclonal antibodies and unlabeled ligands (Sigma Chemical Co) and [3H]-ligands (Amersham International, Cardiff, UK) were used. In subsequent comparative studies, only noradrenaline, A23187, and arachidonate were employed as stimulators due to the limitation of tissue available.
DNA assays were carried out as previously described and validated [16]. Vessel segments in which eicosanoid synthesis had been assessed were weighed and frozen in liquid nitrogen. Tissues were crushed (in liquid nitrogen) in a pestle and mortar and lipids extracted with 3 times 500 µL absolute ethanol. To the residue, 80 µL diaminobenzoic acid was added and tubes incubated at 60°C for 30 minutes. Perchloric acid (800 µL) was then added, vortexed, and centrifuged at 1,000 g for 3 min. Aliquots of supernatant were taken and placed in borosilicate tubes for measurement of transmission in a fluorimeter. DNA concentrations were calculated from a concomitantly prepared standard curve. For each animal, samples of ungrafted saphenous vein, carotid artery, and vein graft were placed in phosphate-buffered formalin for histological appraisal, as previously described [16].
Data Analysis and Statistics
Data are related to micrograms of DNA (rather than protein or wet weight) because this varies between the different vascular tissues. Each data point is expressed as mean ± standard deviation with numbers of observations in parentheses. Statistical comparisons were undertaken using Mann-Whitney rank sum tests.
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Results
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The time course of medial and intimal thickening in this pig venous arterial graft model has been previously reported [16]. Consistent with this previous work, 4 weeks after implantation of grafts, there was an increase in medial thickness as compared with ungrafted vein and a detectable neointima composed of large amounts of extracellular matrix and smooth muscle-like cells. Investigations using specific immunocytochemical markers in four animals indicated the presence of leukocytes in the media of vein grafts but not in ungrafted saphenous veins or carotid arteries (data not shown). Histologic examination also demonstrated that the endothelium remained intact in all tissues and, therefore, that harvesting and flushing procedures elicited no deleterious effects on these cells. The DNA concentration in vein grafts was 2 to 3 times greater per unit weight (wet) than ungrafted saphenous vein, whereas it more closely resembled the DNA/wet weight ratio in carotid arteries. Because all the parameters measured in this study are intracellular, data are related to DNA rather than protein or wet weight.
In characterization studies in carotid arteries, phorbol ester dibutyrate (a protein kinase C activator), sodium fluoride (a nonspecific G protein activator), A23187 (creates artificial calcium channels), thapsigargin (elevates cytosolic calcium), and arachidonate (PGI2 substrate) and the receptor agonists, adrenaline, noradrenaline, and angiotensin II all stimulated PGI2 synthesis in a dose-dependent manner (Figs 1, 2
). Angiotensin I was also a weak stimulator indicating the presence of angiotensin converting enzyme in the carotid artery. These data confirm that PGI2 synthesis in pig blood vessels is controlled in much the same way as in vessels from other species (eg, rats, rabbits, and humans). Given the limitations of tissue availability from any one animal, only three stimulators were used in subsequent studies: noradrenaline, A23187, and arachidonic acid. Both yohimbine and prazosin (alpha adrenoceptor antagonists) but not atenolol or proranolol (beta-adrenoceptor antagonists) inhibited noradrenaline-stimulated PGI2 synthesis, confirming that noradrenaline is acting via specific alpha adrenoceptors.
Synthesis of PGI2 in response to noradrenaline (Fig 3), A23187 (Fig 4), and arachidonate (Fig 5) was markedly diminished in vein grafts compared with either ungrafted saphenous veins or carotid arteries. In turn, PGI2 synthesis in response to noradrenaline (see Fig 3) and A23187 (see Fig 4) was significantly greater in carotid arteries compared to ungrafted saphenous veins, whereas there was no difference in arachidonate-stimulated PGI2 synthesis between the two vessels (see Fig 5).
Release of LTB4 was quantitatively much lower than PGI2 and was barely detectable in carotid arteries and ungrafted saphenous veins (Figs 6, 7). Furthermore, LTB4 synthesis was stimulated by A23187 and arachidonate but not by noradrenaline. A23187- and arachidonate-stimulated LTB4 release was significantly greater in vein grafts compared to ungrafted saphenous vein and carotid artery (see Figs 6, 7).
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Comment
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The present study demonstrates that vasoconstrictors and the intracellular mechanisms governing PGI2 synthesis in porcine blood vessels are similar to those in other species, including humans [5, 10, 15]. The sequence of events can be summarized as activation of receptors, G protein, and phospholipase C; generation of diacyl glycerol (activates PKC) and inositol trisphosphate (elicits an increase in cytosolic Ca2+); activation of PLA2, and generation of arachidonate substrate [5, 9, 10].
Thus, in response to noradrenaline, arachidonic acid, and calcium ionophore A23187, PGI2 release was found to be markedly diminished in vein grafts compared with ungrafted saphenous veins and carotid arteries. Mechanistically, the similarity of the pattern of responses to noradrenaline, A23187, and arachidonate in the vein grafts points to there being no effect of implantation in the arterial system on adrenoceptors or calcium mobilization linked to PGI2, but rather to a reduction of cyclooxygenase or PGI2 synthase activity. Given that PGI2 is an inhibitor of vascular smooth muscle cell proliferation and its synthesis is stimulated by mitogens [5, 6], this reduction in PGI2 production in vein grafts may contribute to the processes associated with neointima formation. Furthermore, a reduction of cyclooxygenase activity would also result in a reduction of PGI2, possibly a more important endogenous inhibitor of proliferation than PGI2 [5–7]. This marked reduction of PGI2 synthesizing capacity would also render the vein graft susceptible to platelet and leukocyte adhesion, infiltration, and the release of their mitogens and, therefore, to thrombosis and atherogenesis. PGI2 (and cyclic adenosine monophosphate) are also associated with other key functions relating to myointimal hyperplasia, including tissue plasminogen activator release, proteoglycan synthesis, metalloproteinase activity, and cholesterol metabolism [5, 6]. The interrelationship between these functions and prostacyclin status in the progression of myointimal proliferation in vein grafts thus warrants further consideration.
In contrast to PGI2, LTB4 release was significantly increased in vein grafts compared with ungrafted saphenous veins or carotid arteries. Although the release of LTB4, in quantitative terms, was much lower than PGI2 in the same samples, it is known that LTB4 stimulates DNA synthesis in cultured rat aortic cells at concentrations as low as 10 fmol/L and thymidine incorporation as low as 10 pmol/L [11, 12]. In contrast, PGI2 inhibits cell proliferation at concentrations in the micromole per liter range [7]. Thus, although there is a quantitative differential between the two eicosanoids, the potency of LTB4 relative to PGI2 could nevertheless tend toward an overall proliferative, migratory, and secretory status in the vein graft. Locally elevated concentrations of LTB4 would also be likely to result in further accumulation of leukocytes, because LTB4 is the most potent chemotactic substance known [17]. It is also of interest that LTB4, although released in response to arachidonic acid and A23187, was not stimulated by noradrenaline, indicating that LTB4 was not derived from smooth muscle cells, an obvious alternative source being leukocytes. In support of this, Angelini and associates [13] demonstrated that leukocytes adhere to and infiltrate vein grafts in the porcine model over the same time course. Angelini and associates, however, do not discount the possibility that lipoxygenase and LTB4 synthase is up-regulated in non-smooth muscle regions of the vein graft, in particular, the intima and endothelium. Establishing whether this is the case will require biochemical analysis of these enzymes in different regions of the graft, which was beyond the scope of the present study.
It is notable that the responses in carotid arteries were greater both quantitatively and in terms of sensitivity, at least in response to noradrenaline and A23187 (but not arachidonate), than ungrafted saphenous vein. This indicates that although arteries and veins possess similar amounts of cyclooxygenase (per unit weight of DNA), calcium is more readily mobilized in arteries than veins (both noradrenaline and A23187 elicit calcium mobilization and therefore the activation of phospholipase A2, which liberates arachidonate from endogenous stores [5, 9]. This is perhaps not surprising because calcium is a prerequisite for vasoconstriction, a predominant function of arteries. Because grafted veins adapt to the hemodynamic conditions of the arterial circulation [18], one would expect some modification of their biochemical behavior. However, in the present study, PGI2 synthesis was found to be markedly reduced in vein grafts compared with both ungrafted saphenous veins and to a far greater extent, carotid arteries. Thus, rather than the implanted vein adapting and becoming ``arterialized,'' it actually becomes less so. This lack of adaptability confirms the limitations of saphenous vein as a bypass conduit. One reason the internal mammary artery graft is protected from myointimal hyperplasia may be the greater PGI2 synthesizing capacity displayed by this vessel compared with saphenous vein [19]. Furthermore, surgical preparation of saphenous veins for use in coronary artery bypass grafting results in a profound reduction in PGI2 synthesizing capacity [20]. This has been identified as a possible contributing factor to early thrombus formation and development of myointimal hyperplasia in vein grafts [20].
In conclusion, there is a marked down-regulation of cyclooxygenase or PGI2 synthase in porcine vein grafts, which may constitute an additional phenotypic change that would augment hyperplastic processes. Furthermore, local increases in LTB4 synthesis (which may also be derived from leukocytes which have infiltrated the graft) may contribute to increased intimal proliferation by direct promitogenic effects on smooth muscle cells. In view of these findings and the protective properties of PGI2, further exploration of the use of PGI2 analogues (eg, iloprost or carabacyclin) and enhancers of cyclic adenosine monophosphate synthesis (eg, phosphodiesterase inhibitors) to reduce early thrombosis and ameliorate myointimal hyperplasia in saphenous vein grafts is warranted. The same principle applies to lipoxygenase inhibitors and LTB4 receptor antagonists.
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Acknowledgments
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This research was supported by the British Heart Foundation and the Wellcome Trust.
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Footnotes
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Address reprint requests to Dr Jeremy, Bristol Heart Institute, Bristol Royal Infirmary, University of Bristol, Bristol BS2 8HW, United Kingdom.
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References
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Top
Abstract
Introduction
), archidonic acid (
), calcium ionophore A23187 (
), and thapsigargin (
). Each point = mean ± standard deviation (n = 10).
). Each point = mean ± standard deviation (n = 8). (
p < 0.001 (SVG versus USV); #p < 0.001 (SVG versus CA); 
sp < 0.001 (USV versus CA).
