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Ann Thorac Surg 2001;72:1290-1297
© 2001 The Society of Thoracic Surgeons
a Department of Cardiovascular Surgery, Centro Cardiologico "I. Monzino" Foundation IRCCS, Milan, Italy
b Department of Pharmacology, Chemotherapy, and Medical Toxicology, University of Milan, Milan, Italy
c Department of Human Anatomy, University of Milan, Milan, Italy
d Department of Pharmacological Sciences, University of Milan, Milan, Italy
Address reprint requests to Dr Pompilio, Department of Cardiovascular Surgery, Centro Cardiologico Monzino Foundation IRCCS, Via Parea 4, 20138 Milan, Italy
e-mail: giulio.pompilio{at}cardiologicomonzino.it
Presented at the Thirty-seventh Annual Meeting of The Society of Thoracic Surgeons, New Orleans, LA, Jan 29–31, 2001.
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Abstract |
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Methods. An IMA segment from 48 selected patients undergoing coronary artery bypass grafting was harvested intraoperatively and assigned to one of four groups (n = 12): diabetics requiring therapy, hypertensives, hypercholesterolemic, and nondiabetic-normotensive-normocholesterolemic patients. Internal mammary artery specimens were cut into rings and suspended in organ bath chambers, and the isometric tension of vascular tissues was recorded. The IMA rings were (1) precontracted with norepinephrine, and the endothelium-derived relaxation was evaluated by cumulative addition of acetylcholine, (2) contracted with cumulative concentrations of endothelin-1, and (3) contracted with the nitric oxide synthase inhibitor, NG-monomethyl-L-arginine. Furthermore, the release of prostacyclin by the IMA rings was directly measured during basal tone conditions and at the end of the various pharmacologic interventions. Histology of IMA rings was randomly performed.
Results. The results obtained in these experiments showed that IMA rings harvested from hypertensive patients have the greatest impairment of endothelium-dependent response to relaxant and contracting stimuli (p < 0.01 versus nondiabetic-normotensive-normocholesterolemic tissues; p < 0.05 versus hypercholesterolemic and diabetic tissues) and prostacyclin release in normal and stimulated conditions. To a lesser extent, hypercholesterolemic and diabetic tissues show similar depression (diabetic > hypercholesterolemic) both of relaxation and prostacyclin production, with respect to nondiabetic-normotensive-normocholesterolemic specimens (p < 0.05). Histology findings (scanning electron microscopy) did not differ in multiple sections from vessel studies.
Conclusions. Major cardiovascular risk factors affect the endothelium-dependent vasoactive homeostasis of human IMA differently. Depression of relaxation is highest in patients with a history of hypertension. These findings may be pertinent to early and long-term treatment of patients undergoing coronary artery bypass grafting.
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Introduction |
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In disease states such as heart failure, diabetes, hypertension, and hypercholesterolemia, the balance between the endothelial production of vasodilating and vasoconstricting factors is abnormal. This alteration of vascular function has been termed endothelial dysfunction [2]. The mechanisms underlying it are almost certainly multifactorial, and seem to be dependent on the specific pathologic condition, its duration, and the vascular bed being studied [3].
The endothelium-dependent relaxation of internal mammary arteries (IMA), the major arterial graft for coronary bypass grafting (CABG), has been widely investigated to understand the biologic basis of its superior patency rates when compared with saphenous vein grafts [4]. Preferential NO-mediated relaxation [5] and PGI2 release [6] appear to make IMA graft resistant to vasoconstriction, development of intimal thickening, and thrombus formation [7]. Nonetheless, impaired endothelial function may affect the patency and function of IMA coronary artery bypass grafts, leading to vasoconstriction, early and late graft failure, and remodeling. Recently, possible mechanisms of IMA-depressed endothelial-dependent relaxation in the presence of cardiovascular risk factors were investigated [8]. However, comparative studies on IMA endothelial-dependent function in patients with known cardiovascular risk factors have not yet been performed. Alterations of the endothelial properties of IMA grafts in the presence of single or combined risk factors may have some relevance in the clinical setting.
The present study provides clear evidence that segments of IMA harvested from selected hypertensive (HT), hypercholesterolemic (HC), and diabetic (D) patients show, in a different manner, a significant impairment of endothelial-dependent relaxation.
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Material and methods |
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TopAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsDiscussionReferences |
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A number of segments of IMA were harvested intraoperatively from 48 selected white male patients undergoing CABG (age range, 47 to 72 years) who had not received antiplatelet drugs for 10 days or angiotensin-converting enzyme inhibitors for at least 3 days. These patients were divided into four groups (n = 12): D requiring therapy, HT, HC, and nondiabetic-normotensive-normocholesterolemic (NNN) patients. Clinical risk factors were categorized as follows: HC defined as total plasma cholesterol level more than 4.8 mmol/L; D defined as current treatment with insulin or oral hypoglycemic agents; and HT defined as current treatment with antihypertensive agents.
Internal mammary artery harvesting and preparation
Human IMA was dissected as a pedicle with its venae comitantes from the thoracic wall by a no-touch technique leaving the vessels surrounded by internal thoracic fascia. No vasodilators were administered systematically or topically during the harvest period. After anticoagulation with heparin, IMAs were checked for optimal flow. If satisfactory, the distal end (1 to 2 cm) was divided and placed in cold (4°C) physiologic salt solution [5]. The vessel segments were immediately transferred to the laboratory.
Isolated internal mammary artery rings
Segments of IMA (n = 12 per group) obtained from the four experimental groups of patients were cleaned in Krebs-Henseleit solution to remove adherent connective tissue and then cut into rings of about 3 to 5 mm in length. The rings were handled carefully to avoid damage to the inner surface and suspended in a 10-mL organ bath chamber containing Krebs-Henseleit solution with the following composition (in mmol/L): NaCl, 118; KCl, 2.8; KH2PO4, 1.2; CaCl2, 2.5; MgSO4, 1.2; NaHCO3, 25; glucose, 11.0; and EDTA, 0.03. The medium was gassed with a mixture of CO2 (5%) and O2 (95%) and maintained at 37°C (pH 7.4). The IMA rings were connected by means of silk sutures to force-displacement transducers (model 7004, U. Basile, Comerio, Varese, Italy), and changes in isometric force were displayed on a Gemini chart recorder (model 7070, U. Basile). All vascular tissues were gradually stretched to a basal resting tension of 1.5 to 2.0 g, which was maintained throughout the experiment. The preparations were allowed to equilibrate for 60 to 80 minutes, and the Krebs-Henseleit solution was changed every 20 minutes. To evaluate maximal contraction, tissues were depolarized with potassium chloride (KCl; 0.1 mole/L) and washed with Krebs-Henseleit solution. After 30 minutes, IMA rings were contracted by norepinephrine (NE, 3 x 10-6 mol/L) and when the contractile response was stabilized (steady-state phase, 13 to 15 minutes), endothelium-dependent relaxation was evaluated by cumulative addition of acetylcholine (Ach, from 10-10 mol/L to 10-4 mol/L). The relaxation capacity of the NO donor sodium nitroprusside (1 x 10-6 mol/L) was recorded at the end of the dose-response curves for Ach. After washout, the same IMA ring preparations were challenged with cumulative concentrations of endothelin-1 (ET-1, from 10-11 mol/L to 10-6 mol/L) and then with NG-monomethyl-L-arginine (L-NMMA, 1 x 10-4 M) [9]. The experiments with the nitric oxide synthase (NOS) inhibitor were performed 20 to 30 minutes after the tone of the IMA rings had returned to baseline.
6-keto-prostaglandin F1
determination
After a suitable period of equilibration of the IMA rings, the basal PGI2-releasing capacity of the tissue was measured by evaluating the concentration of 6-keto-prostaglandin F1 (6-keto-PGF1; the stable metabolite of PGI2) in 1 mL of the bathing fluid after 20 minutes of incubation. 6-Keto-prostaglandin F1 was also determined in 1 ml of medium, removed from the organ bath at the end (approximately 20 minutes) of ET-1 cumulative dose-response curves. The rate of release of 6-keto-PGF1 was determined by enzyme immunoassay (detection limit 3 pg/mL), as described by Pradelles and colleagues [10] and expressed as picograms per milligram wet tissue weight.
Scanning electron microscopy
To assess the presence of an intact endothelial layer in the IMA segments, scanning electron microscopy was randomly performed on freshly isolated specimens. Internal mammary artery samples were cut longitudinally and flat-fixed by pinning onto a plastic surface to avoid curling for 1 hour with 3% glutaraldehyde in 0.12 mol/L phosphate buffer at pH 7.4. The samples were then washed in phosphate buffer, dehydrated through a graded ethanol concentration, and dried by critical point method. They were then examined with a Jeol JSM T-200 scanning electron microscope after sputtering with gold.
Drugs
The following drugs were used: NE chloride, Ach chloride, L-NMMA, ET-1, (Sigma Chemical Company, St. Louis, MO); sodium nitroprusside (Merck, Darmstadt, Germany); and enzyme immunoassay kit for 6-keto-PGF1 determination (Amersham Italia, Milan, Italy).
Statistical analysis
All values in the figures and text are expressed as mean ± standard error of the mean. In each experiment, n is the number of patients from which the IMAs were obtained. The maximum relaxation (Emax) and the negative log molar concentration of a given vasodilator (Ach) exhibiting 25% relaxation (pD2 value) were used to analyze the relaxations. The effective concentration of an agonist (ET-1) causing 50% (EC50 value) of the contraction to KCl was calculated for each IMA ring segment separately and was expressed as the negative log molar concentration. Comparisons among means were performed with one-way and two-way analysis of variance for repeated measures and appropriate post hoc tests (Dunnet’s or Newman-Keuls) when indicated. A p value of less than 0.05 indicates a significant difference.
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Results |
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release (the stable metabolite of PGI2) into the bathing fluid of the IMA rings from the different experimental groups of patients. Internal mammary artery tissues from NNN patients (controls) released consistent amounts of the prostanoid (82.1 ± 5.4 pg/mg wt), whereas the corresponding vascular segments from HT patients exhibited a lower release of 6-keto-PGF1 (32.6 ± 4.1 pg/mg wt; p < 0.01 versus NNN patients). Furthermore, in the organ bath of the IMA rings from HC and D patients, the basal release of the PGI2 metabolite was 57.6 ± 5.6 pg/mg wt and 55.4 ± 4.8 pg/mg wt, respectively (p < 0.05 versus NNN and HT patients; Fig 1).
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from IMA rings of patients in the different experimental-groups. In IMA segments from NNN patients, the addition to the organ bath of cumulative concentrations of ET-1 (from 10-11 mol/L to 10-6 mol/L) increased the tension to 59.8% ± 5.2% of the maximal contraction induced by KCl and prompted the simultaneous release of 6-keto-PGF1 (410 ± 28 pg/mg wt).
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generation was found when IMA segments from HT patients were challenged with ET-1. The contraction induced by ET-1 at its peak was 105.3% ± 4.8% of the maximal contraction caused by KCl (p < 0.001 versus NNN patients), and this event was paralleled by a decreased generation of 6-keto-PGF1 (168 ± 16 pg/mg wt; p < 0.001 versus NNN patients).
To a lesser extent, IMA rings harvested from HC and D patients showed similar hyperreactivity to cumulative concentrations of ET-1 and generation of the PGI2 metabolite. The peak of the vasoconstriction to ET-1 was 80.1% ± 5.1% and 88.8% ± 6.2% of the maximal contraction caused by KCl, respectively (p < 0.05 versus NNN and HT patients), and the corresponding release of 6-keto-PGF1 was 278 ± 25 pg/mg wt and 285 ± 19 pg/mg wt, respectively (p < 0.05 versus NNN and HT patients).
Acetylcholine and NG-monomethyl-
-arginine activity
In these experiments, the response of the IMA rings obtained from HT patients to NE (3 x 10-6 mol/L) was significantly higher (2.28 ± 0.14 g over the resting tension; p < 0.05) than in vascular tissues from NNN patients (1.52 ± 0.11 g over the resting tension). The contractions caused by NE in IMA tissue taken from HC patients (1.78 ± 0.16 g over the resting tension) or from D patients (1.81 ± 0.24 g over the resting tension) were not significantly different from NNN preparations (Fig 3). Exposure to cumulative concentrations of Ach (from 10-10 mol/L to 10-4 mol/L) produced a marked relaxation (93% ± 5%) expressed as percentage of NE-induced contraction of the IMA rings from NNN patients. On the contrary, the sensitivity to Ach of the IMA rings from HT patients was much lower, corresponding only to 35% ± 4% (p < 0.001 versus NNN patients). When IMA tissues from HC and D patients were challenged with Ach, the maximum relaxation effect was 65% ± 5% and 56% ± 5%, respectively (p < 0.01 versus NNN patients and p < 0.05 versus HT patients; Fig 3). In these partially contracted preparations, the addition to the organ bath of sodium nitroprusside (1 x 10-5 mol/L), the endothelium-independent vasodilator, caused full relaxation of the smooth muscles (Fig 4).
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Comment |
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In the early stages of arteriosclerosis, the endothelial function of large conduit and small resistance vessels is impaired. Except for smoking, whose impact on endothelial dysfunction remains somewhat controversial, all known risk factors are associated with endothelial dysfunction of epicardial coronary arteries [12]. Experimental data suggest that endothelial dysfunction precedes overt arteriosclerosis and may represent an important early event predisposing conduit vessels to vasospasm and vasoconstriction [2]. Evidence for this phenomenon has been found in various conditions such as hypercholesterolemia, arteriosclerosis, hypertension, diabetes, and heart failure [13]. In HC rabbits and monkeys, vasorelaxation to Ach is almost absent or changes to vasoconstriction [14]. Similar observations have been made in human coronary circulation, both in the presence of overt arteriosclerosis [15] and of predisposing risk factors [16]. The assessment of coronary endothelium-dependent vasodilator function indicated that a progressive deterioration emerges, including early impairment of Ach-induced vasodilator response, followed by reduced flow-dependent vasodilation [17].
The findings of this study extend these observations to dysfunctional nonatherosclerotic IMAs: We compared, for the first time, the results from selected patients with known cardiovascular risk factors. We selected patients with single risk factors—HT, D, HC—excluding known confounding variables of arterial relaxant function, such as drugs, cigarette smoking, sex, and race [18]. We observed that the presence of a single risk factor such as HT, D, or HC is sufficient to severely impair in vitro endothelium-dependent vasoactive functions of nonatherosclerotic IMAs in various ways. Interestingly, IMA segments harvested from HT patients showed a greater degree of dysfunctional NO-PGI2 homeostasis when compared with D and HC subjects. As there are no previous comparative studies, we can speculate that in elastic vessels such as IMAs, the increased intraluminal blood pressure may adversely affect endothelium-dependent relaxation more than other risk factors.
Huraux and coworkers [8] have recently investigated possible underlying mechanisms of endothelial dysfunction in IMAs from patients with cardiovascular risk factors. They found a marked variability in endothelium-dependent relaxation, particularly to Ach, in the presence of an intact endothelium. However, the authors were not able to correlate and quantify endothelial dysfunction to single risk factors, and attributed some of the variability observed in IMA relaxation to the number of risk factors present. Several mechanisms may be involved in endothelial dysfunction, such as reduced synthesis and release of NO or enhanced activation of NO after its release from endothelial cells by oxygen-derived radicals [19]. However, the precise mechanisms underlying reduced endothelium-dependent vasodilations in the presence of major cardiovascular risk factors such as HT, D, and HC are not understood. It is not clear whether the reduction is caused by a reduced release, an enhanced breakdown, or a reduced response to NO [20–22].
In this study, IMA rings harvested from HT, D, and HC patients exhibited an increased sensitivity to NE and ET-1. These events were also paralleled by the reduction of basal and ET-1–stimulated generation of 6-keto-PGF1, the stable metabolite of PGI2. Moreover, when the same IMA segments were precontracted with NE, Ach—but not the NO donor sodium nitroprusside—significantly lost its relaxant activity. These results suggest that human IMAs harvested from subjects with single cardiovascular risk factors show an impaired NOS activity of their endothelial cells and rule out an alteration of the cyclic guanosine monophosphate effector pathway. Consistent with this view is the increase in tension of IMAs when NOS activity was inhibited by L-NMMA. Thus, hyperresponsiveness to vasoconstrictors would seem to be caused by a reduced formation of NO. These findings agree with recent observations by Hamilton and coworkers [5] on the predominance of an NO pathway in mediating relaxation in human IMAs. As previously described, the contribution to endothelial-dependent relaxation by PGI2 formed in response to vasoconstrictors is not significant in systemic human arteries [23]. Prostacyclin production is directed mainly toward the vascular lumen, so it would have an antiplatelet, not vasodilating, effect [24].
The major functional consequence of impaired NO availability in human vessels is the loss of flow-dependent dilation that may favor vasoconstriction at sites where vasodilation would ordinarily occur in response to an increase in flow [2]. This paradoxical response is because of the reduced or absent NO-mediated shear-induced dilation, which opposes myogenic vasoconstriction [25]. Moreover, the intact endothelium plays an important role in maintaining the balance between proaggregatory and antiaggregatory behavior of platelets, as both NO and PGI2 inhibit platelet aggregation and adhesion. Hence, defective endothelial function predisposes to the deposition of platelets. Competent endothelial homeostasis plays a crucial role in the regulation of leukocyte accumulation in the vessel wall, as well as in the control of smooth muscle proliferation [26], thus preventing intimal hyperplasia and arteriosclerosis.
The present study focuses on pharmacologically induced alterations of IMA endothelial properties in the presence of single risk factors. The issue of a direct measurement of NO production through a membrane-type NO-sensitive electrode has not been addressed [27], and the contribution of NO to endothelium-dependent relaxation was estimated by evaluating the effects of inhibitors of endothelial NO, according to Vanhoutte [18]. Although it has been shown [28] that none of the NOS inhibitors completely blocks NO biosynthesis and release, we believe that a possible underestimation of NO contribution in IMA relaxation does not affect the inferences of this study.
Moreover, although providing insight into how the chronic presence of cardiovascular risk factors may affect endothelial vasoactive function of IMAs, the study may not be applicable to diseased states such as overt arteriosclerosis and to the acute onset of the risk factors considered, which may determine different alterations of the endothelial function of IMAs.
In conclusion, IMAs of patients with HT, D, and HC show an in vitro impaired relaxant and antithrombotic capacity that may facilitate the occurrence of vasoconstriction and thrombus formation, with the consequent release of vasoconstrictor substances that may exacerbate vasospasm [29] and predispose to early and late graft dysfunction or failure. These findings could have some relevance for early and long-term treatment of patients undergoing CABG. In particular, IMAs of HT patients may be prone to early dysfunction after CABG in the absence of specific vasodilators. Comparative clinical and angiographic studies on late patency of IMA graft in the presence of known risk factors should be considered.
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Acknowledgments |
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This study was supported in part by a grant from the Italian Ministry of Health (ICS030.6/RC00.CC01).
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Discussion |
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DR POMPILIO: Certainly it is difficult to predict dysfunction of a mammary artery after implant. I think that sometimes early internal mammary artery graft dysfunction may occur, either vasoconstriction or graft occlusion. Probably some clinical studies should address the correlation between the presence, for example, of hypertension and these early dysfunctional problems.
DR RALPH J. DAMIANO (St. Louis, MO): I have a couple of questions for you. First of all, was there any overlap between the clinical risk factors? At least in this country, we very rarely see someone who is a diabetic who does not also have hypertension or hyperlipidemia. You only had diabetics who were nonhypertensive and nonhyperlipidemic. I wonder whether you could tell us about the separation of risk factors among the different groups.
How much of the effect that you saw may have been related to the drug regimen that the patients were on preoperatively as opposed to any inherent defect in the endothelium? Some of the oral hypoglycemic agents, as you know, block the ATP-sensitive potassium channel, which plays a role in endothelial vasodilation. Certainly, there are a large number of other drugs that may have an effect on the endothelium. Did you look at the preoperative drug regimen and how it may have affected your results?
DR POMPILIO: Thank you for your questions. As far as patient selection is concerned, as I said before, this is a 3-year prospective study. Actually we carefully tried to select patients with a single risk factor, for example, hypertension without diabetes or hypercholesterolemia, and this was our major effort: to select patients for the study. As for your second question, some drugs are very important in altering endothelial function. In this study we made sure that our patients did not take confounding drugs. For instance, antiplatelet drugs were stopped at least 7 days before the operation, or angiotensin-converting enzyme inhibitors at least 3 days before the operation, and so on. We tried to eliminate the problem of drug influence on endothelium-dependent response of the mammary artery.
DR CLINTON E. BAISDEN (Temple, TX): As I understand it, nitric oxide is derived from L-arginine in the presence of nitric oxide synthase and the cofactor, tetrahydrobiopterin, also called BH4. It has been shown that in laboratory animals with diabetes, tetrahydrobiopterin is deficient or absent. As far as I know, tetrahydrobiopterin has never been measured in human endothelial cells, and that is what we attempting in our laboratory with Texas A & M. We are growing a cell line from the endothelium of internal mammary arteries and saphenous veins obtained from patients who have diabetes. I was wondering whether you had any experience from your studies with the co-factor tetrahydrobiopterin as it applies to humans?
DR POMPILIO: Thank you for the question. No, we have no experience, we did not measure this agent in diabetic patients. What we have observed is the final product of several mechanisms of endothelial dysfunction, in particular of nitric oxide synthase activity, because it is well known that in the presence of different risk factors, such as, for example, hypertension or diabetes, the basic mechanisms of endothelial dysfunction are multifactorial. Thus, we have not tried to address this issue. We have observed the final product of what we think is a multifactorial event, which is the inhibition or the impairment of nitric oxide synthase activity.
DR OZ M. SHAPIRA (Boston, MA): Your study population included only men. Did you conduct any tests in women, considering the protective effects of estrogen on endothelial function?
DR POMPILIO: We excluded women because the endothelial-dependent response in vitro is reported to be different from men. So, we do not have experience in women. We try to select only men just to restrict our selection criteria.
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