Ann Thorac Surg 2003;75:1437-1442
© 2003 The Society of Thoracic Surgeons
Original article: cardiovascular
Nitric oxide donating aspirins: novel drugs for the treatment of saphenous vein graft failure
Nilima Shukla, PhDa,
Gianni D. Angelini, FRCSa,
Raimondo Ascione, FRCSa,
Sudath Talpahewa, FRCSa,
Radek Capoun, FRCSa,
Jamie Y. Jeremy, PhDa*
a Bristol Heart Institute, Bristol Royal Infirmary, University of Bristol, Bristol, United Kingdom
Accepted for publication November 22, 2002.
* Address reprint requests to Dr Jeremy, Bristol Heart Institute, Bristol Royal Infirmary, University of Bristol, Bristol BS2 8HW, UK.
e-mail: j.y.jeremy{at}bristol.ac.uk
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Abstract
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BACKGROUND: A new class of nitric oxide donating aspirin (NO-ASA) drugs may increase the therapeutic impact of aspirin in saphenous vein coronary artery bypass grafting (CABG) not only through the inhibition of thrombosis but also through a reduction of vasospasm and inhibition of vascular smooth muscle cell (VSMC) proliferation (effects that are inhibited by NO but not ASA). In order to test this proposal the effect of three NO-ASA drugs (NCX 4040, NCX4050, and NCX4060) on in vitro relaxation and cyclic guanosine monophosphate (cGMP) formation in the human isolated saphenous vein and the proliferation of human VSMCs was investigated.
METHODS: Saphenous vein segments were obtained from 30 patients undergoing CABG (median age, 59 years; range, 49 to 68). The effect of the NO-ASA adducts, ASA alone, and sodium nitroprusside (NO donor) were investigated on (1) relaxation of phenylephrine-stimulated contraction using an organ bath, (2) cyclic guanosine monophosphate (cGMP) formation using an enzyme-linked immunosorbent assay, and (3) the proliferation of VSMCs derived from saphenous vein using bromo-deoxyuridine (BRDU) incorporation.
RESULTS: All three NO-ASA adducts (at concentrations that inhibited responses by 50% [IC50s] between 1 µmol/L and 100 µmol/L) and nitroprusside (at IC50s between 0.5 and 10 µmol/L) elicited relaxation of isolated human saphenous vein, promoted cGMP formation, and inhibited VSMC proliferation whereas ASA alone (up to 100 µmol/L) had no effect on any variable.
CONCLUSIONS: These data indicate that the NO-ASA adducts by virtue of their capacity to release NO and stimulate guanylyl cyclase may be useful not only in the prevention of thrombosis following CABG but also the reduction of saphenous vein graft spasm and neointima formation.
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Introduction
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Autologous saphenous vein graft failure after coronary artery bypass graft surgery (CABG) occurs in as many as 18% of cases within the first week after surgery, due principally to thrombosis [1–5]. Vein graft thickening is the main cause of late failure, which results in an overall failure rate of 50% within 10 years [1–5]. Vein graft thickening is a result of increased medial thickening and neointima formation, both of which involve the proliferation and migration of vascular smooth muscle cells (VSMC) [1, 2, 5]. Superimposed on neointima formation is atherogenesis, which ultimately leads to graft occlusion [1, 2, 5].
Aspirin (ASA) is administered routinely to patients who have undergone CABG because it reduces the incidence of vein graft thrombosis in both the short and long term [3, 4]. Aspirin has no effect on long-term graft thickening however [3, 4]. Aspirin does not inhibit VSMC proliferation [6] nor does it inhibit the adhesion of platelets and leukocytes or the release of prothrombotic and mitogenic substances from these cells [7]. Vascular reconstructive surgery also reduces the potency of ASA [8]. Platelets from patients with vascular disease are also hyperresponsive to agonists and as such resistant to standard doses of ASA [9]. Vasoconstriction of vein grafts immediately after implantation, which promotes thrombosis and neointima formation, is also unaffected by ASA [5, 6].
In contrast to ASA, nitric oxide (NO) is a potent inhibitor of platelet and leukocyte adhesion and the release of mitogens, and VSMC proliferation and is a potent vasodilator [10–12]. There is substantial evidence that NO inhibits neointima formation [10]. It was suggested therefore that the coadministration of a nitric oxide (NO) donor may compensate for the limitations of ASA [5]. A novel drug type that intrinsically fulfills these pharmacologic criteria are the NO-releasing aspirins (NO-ASA) [13]. We therefore studied the effect of three NO-ASA adducts (compared with ASA and the NO donor sodium nitroprusside alone) on the in vitro relaxation and formation of cyclic guanosine monophosphate (cGMP) in human saphenous vein and on the proliferation of VSMCs also derived from human saphenous vein.
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Material and methods
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Tissues
Human saphenous vein segments (surplus samples) were obtained at the end of surgery from a total of 30 patients undergoing CABG, from whom informed consent had been obtained (15 men; median age, 59 years; range, 49 to 68). Vein segments were cut into 2-mm rings for the organ bath and cGMP studies and the remainder used for the preparation of cultured VSMCs (for proliferation and toxicity), as described below. Successive vein segments were used until the target number of tests for each drug had been reached.
Drugs
The NO-ASA adducts studied were NCX 4040 (2-acetoxybenzoate 2-(2-nitroxy)-butyl ester), NCX 4050 (2-acetoxybenzoate 2-(2-nitroxy-methyl)-phenyl ester, and NCX 4060 (2-acetyloxy)benzoic acid 6-(nitrooxymethyl)-2-pyridinylmethyl ester chloride), which were supplied by NiCox SA (Nice, France). Nitroaspirins are stable nitrate-ester compounds that require enzymatic hydrolysis to liberate NO [13]. The kinetics of this metabolic processing leads to durable production of NO released at a constant rate from the site of metabolism [13]. These drugs release NO at different rates owing to their different chemical structures [13].
Organ bath studies
The measurement of relaxation using an organ bath was carried out as previously described [14]. Briefly, adventitia and fatty tissue was removed and the vein cut into 2-mm rings and placed in oxygenated Kreb’s Ringer bicarbonate buffer (KRB) that had the following composition (in mmol/L): NaCl 120, KCl 4.7, MgSO40.7H2O 0.6, KH2PO4 1.2, NaHCO325, CaCl22.5, and glucose 5. The rings were mounted in 20-mL capacity organ baths containing KRB solution gassed with a mixture of 95% O2/5% CO2 to a pH of 7.4. The rings were suspended between two metal hooks one of which was attached thorugh a SS121A force displacement transducer and data were recorded using BioLab software (Lab Prosystems, Norfolk, UK). An initial tension of 3 g was applied to the suspended rings and allowed to equilibrate for at least 30 minutes and then tension reapplied until a stable base line was achieved. This resting tension was found in our hands to be optimal for development of optimal active force [14]. The effect of NX 4040, NCX 4050, NCX 4060, ASA alone, and sodium nitroprusside alone added incrementally from 0.01 to 100 µmol/L on the relaxation of saphenous vein rings maximally precontracted with 1 to 4 µmol/L phenylephrine was then studied. Data are expressed as percent of maximal relaxation (100% representing base line tension).
Formation of cGMP
Segments of human saphenous vein in KRB containing 250 µmol/L isobutylmethylxanthine (IBMX; inhibits endogenous phosphodiesterase activity) were incubated for 15 minutes at 37°C before the addition of NCX 4040, NCX 4050, NCX 4060, ASA, and sodium nitroprusside incrementally from 00.1 to 100 µmol/L. After a further incubation of 20 minutes at 37°C reactions were stopped with the addition of 1 mol/L perchloric acid. Saphenous vein segments were then sonicated (to extract nucleotides) and then neutralized with 1 mol/L KHPO4, diluted in assay buffer and measured using an enzyme-linked immunosorbent assay (ELISA) kit (Amersham Ltd, Buckinghamshire, UK) [15]. Data are expressed as fmoles cGMP generated per unit wet weight of tissue per minute.
Vsmc proliferation
Veins were stripped of adventitia and endothelium, cut into small segments and placed in flasks containing Dulbecco’s modified Eagle medium (DMEM; GIBCO, Paisley, Scotland) supplemented with 10% fetal bovine serum, 100 IU/mL penicillin, and 100 µg/mL streptomycin sulfate (GIBCO) at 37°C in a 95/5% O2/CO2 humidified incubator [16]. When confluent, VSMC proliferation was then assayed using bromodeoxyuridine (BRDU) incorporation using a standard commercial kit (Boehringer-Manheim, Germany). Confluent cells were the passaged using 0.05% trypsin/0.02% EDTA (GIBCO) and subcultured in 96-well plates at a ratio of 1:3. Cells were cultured in 0.4% FCS for 96 hours in order to induce quiescence. The NO-ASA adducts, ASA alone, or sodium nitroprusside alone were then added to the cells and proliferation stimulated with 5% FCS (with a 0.4% FCS subset included as growth-arrested control) and incubated for 48 hours in the humidifier at 37°C. Cells were then washed and fixed with alcohol. Bromodeoxyuridine antigen, 100 µL dye, and sulfuric acid were then added and absorbency at 450 nm of each well measured using a Multiskan R plus version 2.03 and Labsystems Genesis program (Helsinki, Finland) from which cell proliferation was determined. Data are rexpressed as percent inhibition of proliferation: 100% represents BRDU incorporation elicited by 5% FCS and 0% represents incorporation in growth arrested cells (ie, 0.4% FCS).
Lactic acid dehydrogenase
In parallel experiments on cultured VSMCs from the same patients, assays of lactic acid dehydrogenase (LDH) in cell culture medium after exposure to drugs under identical incubation conditions as for BRDU incorporation were also carried out as an index of cytotoxicity using commercial kits (Boehringer-Manheim, Germany) [16]. Vascular smooth muscle cells were seeded into 24-well plates and cultured for 48 hours to reach semiconfluence and rendered quiescent as described above. Vascular smooth muscle cells were treated with the NO-ASA adducts, ASA, and sodium nitroprusside and incubated for a further 48 hours. Supernatants were carefully removed and stored and the monolayer of cells solubilized with 1% Triton X-100 for 1 hour at room temperature. These supernatants and solubilized cells were then processed for measurement of LDH levels. Lactic acid dehydrogenase release was expressed as a percentage of LDH in the supernatant relative to total LDH (LDH in supernatants plus LDH in cells).
Data analysis and statistics
For organ bath, cGMP, and VSMC proliferation studies the responses in rings or cells derived from one individual sample of saphenous vein were carried out in quadruplicate for each drug dose from which a mean value was calculated. For compilation of dose-response curves the data from eight consecutive saphenous vein samples were obtained by calculating the mean (± SEM) of the means obtained above and expressed graphically in Figures 1, 2, and 3 . Data were analyzed using repeated measures analysis of variance (ANOVA). Post hoc analysis between different concentrations were carried out using paired t tests
when the main effect for concentration was significant (p < 0.05). No correlation has been applied to the p values for multiple comparisons
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Fig 1. Effect of nitrated aspirin adducts, NCX4040 (open circles), NCX 4050 (solid triangles), NCX 4060 (squares), aspirin alone (open triangles), and sodium nitroprusside (solid circles) on the relaxation of human saphenous vein in vitro precontracted with 1 to 4 µmol/L phenylephrine. (Each point is mean ± SD, n = 8; M = molar.) *p less than 0.001 comparing control values with drug treatment.
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Fig 2. Effect of NCX4040 (open circles), NCX 4050 (solid triangles), NCX 4060 (squares), aspirin alone (open triangles), and sodium nitroprusside (solid circles) on cyclic guanosine monophosphate (cGMP) formation by human saphenous vein in vitro. (Each point is mean ± SD, n = 8; M = molar.) *p less than 0.001 comparing control values with drug treatment.
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Fig 3. Effect of NCX4040 (open circles), NCX 4050 (solid triangles), NCX 4060 (squares), aspirin alone (open triangles), and sodium nitroprusside (solid circles) on the proliferation of vascular smooth muscle cells (VSMC) obtained from human saphenous vein in vitro. (Each point is mean ± SD, n = 8; M = mean.) *p less than 0.001 comparing control values with drug treatment.
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Results
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NCX 4040, NCX 4050, NCX 4060, and sodium nitroprusside all relaxed human saphenous vein precontracted with phenylephrine in a dose-dependent manner at concentrations that inhibited responses by 50% (IC50s) between 10 and 100 µmol/L, the order of potency being NCX 4040 greater than NCX 4050 greater than NCX 4060 (Fig 1). Nitroprusside also elicited relaxation at an IC50 of 1 µmol/L (Fig 1). In contrast, ASA had no effect on relaxation (Fig 1). In samples from the same saphenous veins, cyclic GMP formation was stimulated by NCX 4040, NCX 4050, NCX 4060 with IC50s between 1 µmol/L and 10 µmol/L, the order of potency being NCX 4040 greater than NCX 4050 greater than NCX 4060 (Fig 2). Sodium nitroprusside also promoted cGMP formation at an IC50 of 0.38 µmol/L (Fig 2). In contrast, ASA alone had no effect on cGMP formation (Fig 2). These data indicate that the NO moiety and not the ASA moiety of the NO-ASA adducts is responsible for the relaxation of the saphenous vein and through a cGMP-mediated mechanism.
Human VSMC proliferation was inhibited in a dose-dependent manner by NCX 4040, NCX 4050, NCX 4060 with IC50s between 1 µmol/L and 10 µmol/L (Fig 3), the order of potency being NCX 4040 greater than NCX 4050 greater than NCX 4060. Sodium nitroprusside also inhibited the proliferation of VSMCs in a dose-dependent manner at an IC50 of 0.85 µmol/L (Fig 3). In contrast, ASA had no effect on VSMC proliferation.
At concentrations of NCX 4040, NCX 4050, NCX 4060 that inhibited VSMC proliferation there were no significant effect on LDH release cells, demonstrating that these drugs had no effect on the viability of the cells and their effects were not due to cytotoxicity. At higher concentrations of ASA and nitroprusside there were cytotoxic effects (Table 1).
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Table 1. Effect of Continual Exposure of Different Drugs on Lactic Acid Dehydrogenase Release (Percent Increase) by Human Vascular Smooth Muscle Cells Cultured for 48 Hours
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Comment
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The effects of the NO-ASA adducts investigated in this study demonstrate that the treatment of vein graft disease is an indication for this drug class by virtue of their capacity to generate NO and activate guanylyl cyclase. All three NO-ASA drugs elicited relaxation and the formation of cGMP on human saphenous vein whereas ASA alone had no effect. Previous studies have demonstrated that nitrated ASA drugs release NO [13]. Thus the lack of effect of ASA and the stimulatory effect on cGMP formation indicates that the relaxation promoted by the NO-ASA adducts is due to the release of NO. Acute constriction of vein grafts, a consequence of surgical preparation, may promote thombosis and impair perfusion of the myocardium immediately after surgery [17]. Indeed the NO-donor glyceryl trinitrate is widely used to induce dilation of vein grafts before implantation [17]. Furthermore a major limitation of ASA is that it also inhibits prostacyclin (PGI2) formation by human vascular tissue, in vivo [18]. PGI2 possesses properties that counter the pathologic sequelae of early vein graft failure including the inhibition of platelet and leukocyte adhesion and dilation of the saphenous vein [6]. As NO essentially possesses the same bioproperties as PGI2 [6] the NO moiety in the NO-ASA adduct would compensate for this limitation of ASA.
The potent inhibition of VSMC proliferation in cells derived from human saphenous vein also demonstrates that the NO-ASA adducts possesses the potential to prevent neointima formation. Neointima formation, which occurs within the first month after graft implantation, involves the proliferation and migration of VSMC from the medial region of the graft to the intima where they continue to proliferate and secrete matrix proteins [5]. Macrophages infiltrate this new layer where they develop into foam cells, the epicentre of the atherosclerotic lesion, which in turn precipitates thrombotic occlusion in as many as 50% of cases within 10 years after CABG [5]. The concensus therefore is that early inhibition of neointima formation would diminish late vein graft failure [5]. It is also now firmly established that ASA administration has no effect on long-term outcome owing to graft thickening [3, 4]. Nitric oxide inhibits VSMC proliferation and migration, however, as well as neointima formation in vein grafts and after balloon injury [10, 11]. Thus the lack of effect of ASA on VSMC proliferation would be compensated for in the NO-ASA adducts through their NO-donating capacity. ASA also inhibits antiproliferative prostaglandins and therefore may actually promote neointima formation [6], again an effect that would be compensated for by the concomitant release of NO by these adducts. In this context ASA inhibits endothelial regrowth in injured vessels, an effect that would exacerbate and prolong the adhesion of platelets and leukocytes [19]. In contrast there are reports that NO promotes endothelial proliferation, in which case the presence of the NO moiety may compensate for the deleterious effect of ASA alone [10].
Many studies have now established that NO donors prevent the progression of atherogenesis, which is axiomatic in late vein graft failure [10]. A key property of NO is that it inhibits the expression of adhesion molecules (eg, the selectins, GPIIbIIIa, ICAM, and VCAM) in platelets, neutrophils, and in vascular tissue and therefore the adhesion of platelets and leukocytes to vascular tissues [11, 12]. The NO-ASA adducts have been shown to inhibit adhesion molecule expression through a cGMP-dependent mechanism [13, 20]. The surgical preparation of human saphenous vein for bypass grafting involves exposure, dissection, intraluminal distension, side branch ligation, and storage in physiologic solution and invariably causes a high degree of damage to the endothelium [21–23]. That results not only in instant platelet adhesion but also a loss of endothelial-derived NO, which further augments adhesion and vasospasm. Not only platelets but also neutrophils and monocytes have been shown to adhere to the walls of recently implanted vein grafts [5, 21–23]. Apart from thrombosis, the adhesion of platelets and leukocytes have been implicated in both vasospasm and neointima formation because they release a battery of constrictors including serotonin and leukotrienes and mitogens such as platelet-derived growth factor and superoxide [24]. Although ASA has no effect on adhesion molecule expression or on the release reactions of these cells, NO is a potent inhibitor of these events [11, 12, 24]. It follows that the NO-ASA adducts possess the potential to inhibit the pathologic events associated with adherant blood cells, in particular thrombosis vasospasm and the promotion of neointima formation.
To summarize, the present study indicates that NO-ASA adducts constitute a potentially valuable class of drugs in the treatment of both early and late vein graft failure by virtue of possessing two powerful pharmacological moieties: ASA and NO. The NO moiety confers several additional properties that not only compensate for the limitations of ASA but also elicits beneficial effects in its own right. Taken together the repertoire of properties demonstrated by the adducts make them potentially useful not only in treating vein graft disease but also thrombosis and restenosis after percutaneous coronary artery balloon angioplasty. It is also worth noting that the NO moiety of NO-ASA adducts prevents gastric mucosal irritation and erosion, a problem typically associated with the administration of ASA alone [25]. This is a valuable attribute of the adducts because patients who have undergone CABG treatment receive ASA over long periods after the procedure [1].
In conclusion, the various properties of the NO-donating ASA adducts render them potentially valuable in treating the various "ASA-resistent" complications associated with CABG. It is also encouraging that in a preliminary study we found that NCX 4016 is effective in reducing intimal hyperplasia in a pig vein graft model [26]. To determine whether these drugs are clinically effective, the next step will be to compare the antiplatelet efficacy (including cGMP levels, adhesion molecule expression) of an NO-donating aspirin with that of aspirin alone. To determine whether these drugs exert an impact in vivo in human, forearm blood flow would be a convenient and reliable method of assessment. Should these studies prove that the ASA-NO adducts are superior to aspirin, then a long-term clinical trial on vein graft patency would be warranted.
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Acknowledgments
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We are thankful to NiCox SA and the British Heart Foundation for financial support.
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References
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- Mortwani J.G., Topol E.J. Aortocoronary saphenous vein graft disease. Pathogenesis, predisposition and prevention. Circulation 1998;97:916-931.[Abstract/Free Full Text]
- Favaloro R. Critical analysis of coronary artery bypass graft surgery: a 30 year journey. J Am Coll Cardiol 1998;31:1B-63B.
- Goldman S., Copeland J., Moritz T., et al. Saphenous vein graft patency one year after coronary artery bypass surgery and effect of antiplatelet therapy. Circulation 1989;80:1190-1197.[Abstract/Free Full Text]
- Goldman S., Copeland J., Moritz T., et al. Long term graft patency (3 years) after coronary artery surgery. Effects of aspirin: results of a VA co-operative study. Circulation 1994;89:1138-1143.[Abstract/Free Full Text]
- Jeremy J.Y., Mehta D., Bryan A.J., et al. Platelets and saphenous vein graft failure following coronary artery bypass graft surgery. Platelets 1997;8:295-309.
- Jeremy J.Y., Jackson C.L., Bryan A.J., Angelini G.D. Eicosanoids, fatty acids and restenosis following coronary artery bypass graft surgery and balloon angioplasty. Prostagl Leukotr Essential Fatty Acids 1996;54:385-402.[Medline]
- Chronos N.A.F., Wilson D.J., Janes S.L., et al. Aspirin does not effect the flow cytometry detection of fibrinogen binding to or release of
-granules or lysosomes from human platelets. Clin Sci 1994;87:575-580.[Medline]
- Walters T.K., Mitchell D.C., Wood R.F.M. Low-dose aspirin fails to inhibit increased platelet reactivity in patients with peripheral vascular disease. Br J Surg 1993;80:1266-1268.[Medline]
- Barradas M.A., Stansby G., Hamilton G., Mikhailidis D.P. Diminished platelet yield and enhanced platelet aggregability in platelet rich plasma of peripheral vascular disease patients. Int Angiol 1994;13:201-207.
- Jeremy J.Y., Rowe D., Emsley A.M., Newby A.C. Nitric oxide and vascular smooth muscle cell proliferation. Cardiovasc Res 1999;43:658-665.[Abstract/Free Full Text]
- Napoli C., Ignarro L.J. Nitric oxide and atherosclerosis. Nitric Oxide 2001;5:88-97.[Medline]
- Riddell D.R., Owen J.S. Nitric oxide and platelet aggregation. Vitam Hormone 1999;57:25-48.
- Del Soldato P., Sorrentino R., Pinto A. NO-aspirins: a class of new anti-inflammatory and anti-thrombotic agents. Trends Pharmacol Sci 1999;20:319-323.[Medline]
- Aldosoro M., Medina P., Vila J.M., et al. Endothelium-dependent relaxation of human saphenous veins in response to vasopressin, and desmopressin. J Vasc Surg 1997;25:696-703.[Medline]
- Jeremy J.Y., Birkett S., Bryan A.J., Angelini G.D. Cyclic nucleotide synthesis by freshly isolated and surgically prepared human saphenous vein before and after 14 days culture. Eur J Vasc Endovasc Surg 1997;13:72-78.[Medline]
- Shukla N., Jeremy J.Y., Nicholl P., et al. Short term exposure to low concentrations of thapsigargin inhibits [3H]-thymidine incorporation by cultured human vascular smooth muscle cells. Br J Surg 1997;84:325-330.[Medline]
- Chanda J., Canver C.C. Reversal of pre-existing vasospasm in coronary artery conduits. Ann Thorac Surg 2001;72:476-480.[Abstract/Free Full Text]
- Tsang V., Jeremy J.Y., Mikhailidis D.P., et al. The release of prostacyclin by the human aorta. Cardiovasc Res 1988;22:489-493.[Medline]
- Bomberger R.A., DePalma R.B., Ambrose T.A., Manalo P. Aspirin and dipyridamole inhibit endothelial healing. Arch Surg 1982;117:1459-1464.[Abstract]
- Jeremy JY, Napoli C. The limitations of aspirin in the treatment of cardiovascular diseases: development of nitrated aspirin and its applications. Pharmacol Ther 2002 (in press)
- Angelini G.D., Breckenridge H.M., Henderson A.H., Newby A.C. Preparation of human saphenous vein for coronary artery bypass grafting impairs its capacity to produce prostacyclin. Cardiovasc Res 1987;21:28-33.[Medline]
- Angelini G.D., Passani S.L., Breckenridge I.M., Newby A.C. Nature and pressure dependence of damage induced by distension of human saphenous vein coronary artery bypass grafts. Cardiovasc Res 1987;21:902-907.[Medline]
- Angelini G.D., Bryan A.J., Williams H.M.J., et al. Distension promotes platelet and leucocyte adhesion and reduces short term patency in pig arteriovenous bypass grafts. J Cardiothorac Surg 1990;99:4333-4339.
- Jeremy JY, Yim AP, Wan S, Angelini GD. Oxidant stress, nitric oxide and vascular disease. J Cardiovasc Surg 2002 (in press)
- Wallace J.L., Muscara M.N. Nitric oxide releasing NSAIDs: GI-safe antithrombotics. Drugs 1999;2:321-326.
- Wan S, Yim A, Bulbulia R, et al. Nitrated aspirin (NCX 4016) inhibits neointimal formation in porcine vein grafts. Fund Clin Pharmacol 2001;15:126(9P084)
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Abstract
Introduction
