Internal Thoracic Artery Rings Research Articles

Antispastic Effect of Fasudil and Cocktail of Fasudil and Nitroglycerin in Internal Thoracic Artery

BackgroundSpasm of arterial grafts in coronary artery bypass grafting is a clinical problem and can occasionally be lethal. Perioperative spasm in the internal thoracic artery (ITA) and coronary arteries occurs in 0.43% of patients. This study aimed to investigate the antispastic effect of a RhoA/Rho-kinase (Rho-associated coiled-coil-containing protein kinase [ROCK]) inhibitor (fasudil) with and without nitroglycerin in combination in the ITA. MethodsIsolated human ITA rings taken from 68 patients who were undergoing coronary bypass were studied in a myograph. Cumulative concentration-relaxation curves for fasudil (−9 to −3.5 log M) were established in the ITA, which was precontracted with potassium chloride or U46619. The inhibitory effect of fasudil (−6.3 or −5.3 log M) or fasudil in combination with nitroglycerin were also tested. The ROCK2 protein was measured by Western blot. ResultsFasudil caused similar relaxation in ITA rings contracted by potassium chloride or U46619. Pretreatment with −5.3 log M fasudil significantly depressed contraction induced by potassium chloride (P = .004 vs control; P = .017 vs −6.3 log M) and U46619 (P = .010 vs control; P = .041 vs. −6.3 log M). Fasudil in combination with nitroglycerin had more effect and more rapid and sustained relaxation than either vasodilator alone. Fasudil caused a decrease of ROCK2 protein content (P = .014). ConclusionsFasudil fully relaxes some vasoconstrictor-induced contraction and decreases ROCK2 protein content in the ITA. The combination of fasudil and nitroglycerin has a superior effect than either vasodilator alone. The new cocktail solution composed of fasudil and nitroglycerin (pH 7.4) has effective antispastic action and may prove to be a new antispastic method for arterial conduits during coronary bypass surgery.

The sources of calcium for noradrenaline-induced contraction in the human thoracic internal artery.

The aim of the present study was to examine the contribution of intracellular and extracellular calcium sources in contraction caused by noradrenaline (NA) of the human internal thoracic artery (ITA) in vitro. Distal segments of ITA were obtained from 20 patients (aged 38-73, at the time of routine coronary artery surgical revascularization (CABG)). Contractile responses to 10-6mol/L NA in the physiological salt solution and in Ca2+-free solution without and after incubation with 10-6mol/L thapsigargin (TSG) were recorded under isometric conditions. Responses of ITA rings to 1μM NA without incubation with TSG accounted (% of reaction to 80mM KCl) 224.70±14.06% in PSS solution, 141.30±8.66% in Ca2+-free solution, and 80.03±1.71% after PSS restoration and were statistically significantly different (p<0.0001, one-way ANOVA). Responses of ITA rings to 1μM NA with 1μM TSG accounted (% of reaction to 80mM KCl) 114.50±2.79% in Ca2+-free solution and 36.70±2.38% after PSS restoration. Responses in Ca2+-free solution and after PSS restoration without and with TSG were statistically significantly different (p=0.0257 and p<0.0001, respectively-t test). ITA contraction is caused by calcium derived not only from the SR and the extracellular matrix. The delivery of calcium to the space surrounding tissue does not immediately deliver calcium to the myofilaments.

What is the source of anticontractile factor released by the pedicle of human internal thoracic artery?

Perivascular tissue (PVT) surrounding human internal thoracic artery (ITA) releases an unidentified anticontractile factor. The exact source of perivascular tissue-derived relaxing factor (PVRF) is unknown, although the adventitia and adipose tissue have both been suggested as primary candidates, hence the name adventitia or adipocyte-derived relaxing factor (ADRF). To look for the source of ADRF, we examined the dilatory response of human ITA to PVT aliquots in their histological composition. We studied isolated ITA segments from 20 patients subjected to coronary artery surgery. The vessels were skeletonized in vitro. ITA rings and PVT were incubated in separate isolated organ baths. The arterial rings were suspended on stainless steel wire hooks in the organ bath chamber. Vessel wall tension was measured with an isometric force transducer. Skeletonized ITA segments were precontracted with 10(-5.5) M phenylephrine. The 5 ml PVT aliquots were next transferred to the ITA tissue bath, resulting in its relaxation. Subsequently, the whole PVT used during experiment was fixed in paraformaldehyde and subjected to histological examination. Tissue was paraffin-embedded, sectioned and stained with haematoxylin and eosin. The paraffin blocks containing PVT were cut into slices every 800 μm to create three-dimensional model. Every PVT specimen was evaluated morphometrically using the Image Pro Plus software to assess the content of three basic kinds of tissues. The ITA relaxation to PVT aliquots was correlated to the histological composition of the PVT. Phenylephrine elicited a 37.82 mN (Q1 = 26.49; Q3 = 46.31) contraction of the ITA. The addition of PVT aliquots to the skeletonized ITA induced a 54.17% (Q1 = 16.73; Q3 = 68.21) relaxation. The median PVT weight was 786 mg (Q1 = 562; Q3 = 976). The PVT composition was as follows: 30.5% (Q1 = 18.5; Q3 = 55.2) adipose tissue, 53.5 (Q1 = 24.6; Q3 = 66.5) muscular tissue and 13.5% (Q1 = 9.9; Q3 = 20.0) connective tissue. This translated into 197.7 mg of adipose tissue (Q1 = 142.2; Q3 = 393.2), 378.9 mg (Q1 = 178.8; Q3 = 537.0) of muscular tissue and 92.4 mg (Q1 = 68.6; Q3 = 185.8) of connective tissue. Neither PVT mass (r = 0.2, P = 0.92) nor adipose tissue (r = -0.2, P = 0.34), muscular tissue (r = 0.3, P = 0.18) or connective tissue (r = -0.2, P = 0.41) content correlated with ITA relaxation response to PVT aliquots. Adipose tissue from the pedicled ITA graft is an unlikely source of ADRF.

Endothelial nitric oxide synthase enhancer for protection of endothelial function from asymmetric dimethylarginine–induced injury in human internal thoracic artery

Endogenous nitric oxide synthase inhibitor asymmetric dimethylarginine is a cardiovascular risk factor that is elevated in patients with coronary artery disease. We hypothesized that novel endothelial nitric oxide synthase enhancer AVE3085 might improve the endothelial function altered by asymmetric dimethylarginine in the human internal thoracic artery. Cumulative concentration-relaxation curves to acetylcholine (-11 to -5 log mol/L) were established in left internal thoracic artery rings (n = 65) from 27 patients undergoing coronary artery bypass grafting in precontraction induced by U46619 (-8 log mol/L) in the absence or presence of asymmetric dimethylarginine (100 μmol/L) or AVE3085 (30 μmol/L). Protein expressions of endothelial nitric oxide synthase and levels of superoxide anion production were detected. Maximal relaxation induced by acetylcholine was significantly attenuated by asymmetric dimethylarginine (12.7% ± 2.3% vs 35.3% ± 5.0% in control; P < .05) and significantly restored by AVE3085 (23.4% ± 2.8%; P < .05). AVE3085 also markedly restored endothelial nitric oxide synthase expression (0.29 ± 0.008; P = .012) reduced by asymmetric dimethylarginine (0.05 ± 0.04 vs 0.36 ± 0.03 in control; P = .014). Increased superoxide anion production by asymmetric dimethylarginine (2.97 ± 0.25 vs 0.51 ± 0.10 relative light units/[s/mg] in control; P < .05) was inhibited by AVE3805 (0.62 ± 0.104 relative light units/[s/mg]; P < .05). AVE3085 may restore endothelium-dependent relaxation reduced by asymmetric dimethylarginine through upregulation of endothelial nitric oxide synthase expression and inhibition of production of superoxide anion in human internal thoracic artery. These findings provide new insights into endothelial protection of coronary bypass grafting vessels to improve long-term patency of grafts.

The role of nitric oxide and potassium channels in the effect of adrenomedullin in human internal thoracic arteries

We investigated the effects of adrenomedullin (ADM) and the role(s) of cyclooxygenase, nitric oxide (NO) synthase and potassium channels in the effects of ADM in human internal thoracic artery (ITA) rings. Samples of redundant ITA rings were suspended in organ baths and isometric tension was continuously recorded. ADM (10(-10)-10(-7)M) produced concentration-dependent relaxation responses in ITA rings precontracted by phenylephrine. The relaxant responses to ADM were significantly higher in endothelium-intact than denuded preparations. Incubation of ITA rings with indomethacin (10(-5)M) did not cause a significant decrease in relaxant responses to ADM, while 10(-4)M of N(omega)-nitro-L-arginine methyl ester caused a significant decrease. Both specific guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (5x10(-5)M) and ADM receptor antagonist ADM((22-52)) (10(-7)M) also caused significant decreases in relaxant responses to ADM. Neither 4-aminopyridine (5mM) nor glibenclamide (10(-5)M) caused significant alterations in vasodilatory effect of ADM. ADM-induced relaxation was significantly blunted by both charybdotoxin and apamin. The present study provided pharmacological evidence about the functional relaxant effect of ADM in human ITA preparations. The findings suggested that both Ca(2+)-activated potassium channels and endothelium, through release of NO play a major role in ADM-induced relaxations in isolated human ITA preparations.

Reactive oxygen species mediate functional differences in human radial and internal thoracic arteries from smokers

Smoking not only increases the risk that coronary heart disease will develop but also morbidity and mortality in patients with known coronary atherosclerosis and after coronary artery bypass grafting. Excessive generation of reactive oxygen species (ROS) has been implicated as the final common pathway for the development of endothelial dysfunction in various cardiovascular risk factors. This study assessed the influence of smoking on two different human arteries routinely used as coronary artery bypass graft conduits. Isometric tension was recorded on discarded segments of human left internal thoracic artery (ITA) and the radial artery (RA) from smokers and nonsmokers. The contractile response to endothelin-1 was significantly stronger in arteries from smokers than in those from nonsmokers. By contrast, endothelium-dependent relaxant responses to acetylcholine were attenuated in RA rings but enhanced in ITA rings from smokers. In additional experiments, 5-(&6)-chloromethyl-2'-7'-dichlorodihydro-fluorescein diacetate (DCDHF) was used to photochemically detect ROS by confocal imaging of intact ITA and RA. Enhanced production of ROS was induced by exposure of tissues to 28 degrees C. While during exposure to 28 degrees C, basal fluorescence emission was unchanged in ITA rings, it increased significantly in RA rings, indicating enhanced formation of ROS in this peripheral artery. Data suggest that smoking induces endothelial dysfunction by increasing vascular ROS production. Different levels of endogenous antioxidant enzyme activities and the degree of atherosclerotic changes might modulate physiologic and pharmacologic vasoreactivity and be responsible for decreased graft patency of RA compared with ITA conduits, especially in active smokers.

Potential role of vasomotor effects of fibrinogen in bradykinin-induced angioedema

Although bradykinin is known to play a major role in the pathophysiology of hereditary and angiotensin-converting enzyme inhibitor (ACEi)-induced angioedema, other factors acting as triggers or enhancers are likely important as well. We hypothesized that fibrinogen might contribute to ACEi-induced angioedema (eg, through direct actions on vascular tone). Plasma levels of fibrinogen were determined in 59 patients with acute angioedema. Vascular activity of human and bovine fibrinogen and its effects on bradykinin-induced vasodilation and phosphorylation of vasodilator-stimulated phosphoprotein were investigated in small (0.8-1.4 mm in diameter) porcine coronary artery and human internal thoracic artery (ITA) segments. In patients with ACEi-induced angioedema, fibrinogen levels (481 +/- 22 mg/dL, n = 39) were significantly higher than in patients with idiopathic angioedema (302 +/- 15 mg/dL, P < .001). Fibrinogen (1-15 mumol/L) induced a concentration-dependent vasodilation in preconstricted small porcine coronary arteries (n = 13), reaching a maximum vasodilator effect of 70% +/- 4.7%. Likewise, fibrinogen induced a 52.1% +/- 9.1% (n = 7) vasodilation in ITA rings. Fibrinogen vasorelaxations were completely inhibited by abciximab and diminished by endothelial denudation and treatment with the nitric oxide synthase inhibitor L-nitroargininemethylester and glibenclamide (P < .01). Importantly, fibrinogen increased the vasodilator potency of bradykinin by 10-fold (P < .0001) and increased bradykinin-induced vasodilator-stimulated phosphoprotein phosphorylation (P < .01). The increase of plasma fibrinogen levels, its vasodilator activity in human ITAs, and the potentiation of bradykinin-induced vasodilation suggest that fibrinogen might contribute to the pathophysiology of ACEi-induced angioedema. Thus acute-phase proteins, such as fibrinogen, might be viewed as risk factors for bradykinin-induced angioedema.

Perivascular tissue of internal thoracic artery releases potent nitric oxide and prostacyclin-independent anticontractile factor☆

It has been recently suggested that perivascular tissue (PVT) releases hypothetic adipocyte- or adventitia-derived relaxing factor. The aim of the study was to assess anticontractile properties of perivascular tissue of human internal thoracic artery (ITA) and to check if this activity is nitric oxide (NO)- or prostacyclin-dependent. We also analyzed the influence of pleural adipose tissue on ITA reactivity. Human ITA rings were studied in vitro. First, skeletonized and pedicled ITA reactivity to serotonin and angiotensin II was compared. In subsequent experiments fragments of ITA were skeletonized and divided into two preparations. One was incubated alone, the other together with PVT or pleural adipose tissue floating freely in the bath. First, concentration-response curves to either serotonin or angiotensin II were constructed. Tissue was then transferred from one bath to the other and concentration-response curves were reconstructed. The same protocol was applied with the inhibition of NO synthase with L-NMMA (10(-4)M) and cyclooxygenase with indomethacin (10(-5)M). Skeletonization augmented contractile response to serotonin (E(max) 16.6+/-1.85 mN vs 43.8+/-3.87 mN; pedicled vs skeletonized ITA, respectively; p<0.001) and angiotensin II (E(max) 10.9+/-1.07 mN vs 26.6+/-1.45 mN, respectively; p<0.001). PVT presence in the bath caused decrease of E(max) from 40.8+/-5.01 to 20.1+/-2.69 mN for serotonin; p<0.001 and from 31.4+/-3.75 to 13.0+/-1.60 mN for angiotensin II, p<0.001 (PVT(-) vs PVT(+), respectively). PVT did not change ITA sensitivity (EC(50)) to serotonin or angiotensin II. Pleural adipose tissue did not change the contractile response of ITA to serotonin (E(max) 37.2+/-4.95 mN vs 36.3+/-4.83 mN, pleural fat+and pleural fat-, respectively; p=0.9). NO and prostacyclin inhibition failed to abolish anticontractile properties of perivascular tissue. PVT with cyclooxygenase and NO synthase inhibition decreased E(max) of serotonin from 46.6+/-3.03 to 28.2+/-4.02 mN, p<0.001 and E(max) of angiotensin II from 27.2+/-2.00 to 16.4+/-2.10 mN, p<0.001. Perivascular tissue of ITA releases potent, soluble, nitric oxide and prostacyclin-independent anticontractile factor. The pleural adipose tissue does not influence ITA reactivity to vasoconstrictors. Preservation of perivascular tissue may protect against vasospasm of ITA graft in clinical settings.

The Effect of Hydrogen Peroxide in Human Internal Thoracic Arteries: Role of Potassium Channels, Nitric Oxide and Cyclooxygenase Products

We investigated both the effect and the role(s) of potassium channels, nitric oxide (NO) and cyclooxygenase (COX) products in the effect of hydrogen peroxide (H(2)O(2)) in human internal thoracic artery (ITA) rings. Samples of redundant ITA obtained from patients undergoing a coronary artery bypass graft surgery were cut into 3 mm wide rings and suspended in 20 ml organ baths. Isometric tension was continuously measured with an isometric force transducer connected to a computer-based data acquisition system. H(2)O(2) (10(-7)-10(-4) M) produced concentration-dependent relaxation responses in human ITA precontracted by phenylephrine. The relaxant responses to H(2)O(2) did not differ significantly between endothelium-intact and endothelium-denuded preparations. Incubation of human ITA rings with superoxide dismutase (50 U/ml) did not affect the relaxant responses to H(2)O(2), while 1,000 U/ml catalase caused a significant decrease. Incubation of endothelium-intact or endothelium-denuded human ITA rings with voltage-dependent potassium channel blocker 4-aminopyridine (5 mM) significantly inhibited the relaxant responses to H(2)O(2). COX inhibitor indomethacin (10(-5) M) also caused a significant inhibition. Incubation with ATP-dependent potassium channel blocker glibenclamide (10(-6) M) or Ca(2+)-activated potassium channel blocker iberiotoxin (10(-7) M) or NO synthase (NOS) blocker N(omega)-nitro-L: -arginine methyl ester (10(-4) M) did not alter relaxant responses of ITA rings to H(2)O(2). The findings of the present study suggested that H(2)O(2)-induced relaxation responses in human ITA were neither dependant on the endothelium nor blocked by NOS inhibition but they rather seem to depend on the activation of voltage-dependent potassium channels and COX.

The role of potassium channels in the vasodilatory effect of levosimendan in human internal thoracic arteries

We investigated the role of potassium channels in vasodilatory effect of levosimendan in human internal thoracic arteries. Samples of redundant internal thoracic arteries obtained from patients undergoing a coronary artery bypass graft surgery were cut into 3 mm wide rings and suspended in 20 ml organ baths. Isometric tension was continuously measured with an isometric force transducer connected to a computer-based data acquisition system. Levosimendan (10(-8)-10(-5) M) or cromakalim (10(-8)-10(-5) M) produced concentration-dependent relaxation responses in human internal thoracic arteries precontracted by 10(-6) M phenylephrine. The relaxant responses to levosimendan did not differ significantly between endothelium-intact and endothelium-denuded preparations. Incubation of human internal thoracic artery rings with adenosine 3',5'-triphosphate (ATP)-dependent potassium channel blocker glibenclamide (10(-6) M) for 30 min significantly inhibited the relaxant responses to both levosimendan and cromakalim. The Ca2+-activated potassium channel blocker iberiotoxin (10(-7) M) also caused a significant but smaller inhibition on relaxant responses to levosimendan. Incubation of the rings with the voltage-dependent potassium channel blocker 4-aminopyridine (5 mM) for 10 min did not cause significant alterations in relaxant responses to levosimendan. The findings of this study suggested that levosimendan-induced relaxation responses in human internal thoracic arteries were depended on the activation of ATP-dependent and Ca2+-activated potassium channels.

Vascular-wall remodeling of 3 human bypass vessels: Organ culture and smooth muscle cell properties

Late graft occlusions after coronary artery bypass grafting have been ascribed to neointimal hyperplasia. Given the pivotal role of smooth muscle cells in the pathogenesis of neointimal hyperplasia and the phenotypic heterogeneity of smooth muscle cells across vessels, we hypothesized that differences in long-term graft patency are at least partly related to differences in smooth muscle cell properties. The aim of the present study was to compare the vascular-wall remodeling of human internal thoracic artery, radial artery, and saphenous vein bypass conduits. We evaluated the intimal thickening of the human graft segments in organ cultures (histopathology, morphometric, and immunofluorescence analyses) and assessed the properties of cultured smooth muscle cells isolated from these vessels in terms of cell proliferation (tritiated thymidine incorporation), migration (modified Boyden chamber), and collagen synthesis (tritiated proline incorporation). The total vessel-wall growth index and the intimal growth index were significantly higher for saphenous vein rings than for radial artery and internal thoracic artery rings. Immunofluorescence analyses showed predominant involvement of smooth muscle cells in neointimal growth induced by organ culture of saphenous vein rings. Cell proliferation was significantly higher in saphenous vein smooth muscle cells than in radial artery smooth muscle cells and significantly higher in radial artery smooth muscle cells than in internal thoracic artery smooth muscle cells. Migration of smooth muscle cells from saphenous vein grafts was significantly greater than from internal thoracic artery or radial artery grafts. Collagen synthesis was similar in smooth muscle cells from internal thoracic artery, radial artery, and saphenous vein grafts. Ex vivo vascular-wall remodeling and smooth muscle cell intrinsic growth and migratory properties are dissimilar between arterial and venous grafts and might shed light on reported angiographic patency rates of these grafts.

Vascular Function and Mild Renal Impairment in Stable Coronary Artery Disease

In patients with coronary artery disease, the concomitant presence of renal function impairment is associated with decreased survival. We aimed to assess whether in coronary artery diseased patients renal function impairment is associated with systemic vascular function, functional parameters of the renin-angiotensin system, or inflammation as potential mediators for cardiovascular risk. We studied 125 patients, 87% male, with a mean age of 62.2+/-8.2 years; 72% had 3-vessel disease, and mean renal function was 74+/-13 mL/min per 1.73 m2. Internal thoracic artery rings were sampled during coronary bypass surgery and used for in vitro vascular measurements. We could not establish an association between endothelium-dependent vasorelaxation (response to methacholine) and renal function. In addition, vascular response to potassium chloride, phenylephrine, and angiotensin II were not associated with renal function. Finally, serum angiotensin-converting enzyme (ACE) activity, usage of ACE inhibitors, C-reactive protein, and soluble intercellular adhesion molecule 1 were not related to renal function. In coronary artery disease patients, mild renal function impairment is not associated with systemic vasomotor responsiveness, inflammation, or functional systemic parameters of the renin-angiotensin system. The relation between systemic endothelial dysfunction and mild renal insufficiency might be more complicated than previously thought.

Angiotensin-converting enzyme inhibitors reveal non-NO-, non-prostacycline-mediated endothelium-dependent relaxation in internal thoracic artery of hypertensive patients

Background We have shown that treatment of hypertension with ACE inhibitors (ACE-I) enhances relaxation to acetylcholine in human internal thoracic artery (ITA) above this in nonhypertensive patients receiving no ACE-I. Present study assesses the endothelium-dependent responses mediated by neither NO nor prostacyclin in human ITA. Methods We compared isolated ITA rings from hypertensive patients treated with ACE-I (ACE-I group) with those from normotensive patients on no ACE-I (control group). Relaxation to acetylcholine was assessed before and after inhibition of NO synthase and cyclooxygenase with l-NMMA and indomethacin, respectively. Results The maximal relaxation in ACE-I group was 79±3.3% and was depressed by incubation with l-NMMA and indomethacin to 41±2.7% ( p<0.001); pD 2=7.7±0.1 vs. 7.4±0.8 ( p=0.265). The maximal relaxation to acetylcholine was lower in the control group: 65±3.3% ( p=0.01); pD 2=7.5±0.1 ( p=0.07). Incubation with l-NMMA and indomethacin produced contraction to acetylcholine with a maximum of 43±7% ( p<0.001); pD 2=5.3±0.3 ( p<0.001). The area under the concentration–response curve for acetylcholine-induced relaxation in ACE-I group equaled [arbitrary units] 596±71 and after incubation with l-NMMA and indomethacin 281±40 ( p=0.002). Estimated LNMMA- and indomethacin-resistant relaxation, absent in control group, accounted for 47±4% of relaxation to acetylcholine in ACE-I group. Estimated NO- and prostacyclin-mediated relaxation was higher in control group than ACE-I group: 628±74 vs. 315±47 ( p=0.009). Conclusions The results suggest that therapy with ACE-I improves endothelial function of hypertensive patients mainly by enhancing the endothelium-derived hyperpolarizing factor (EDHF) (and not NO)-mediated responses. It seems that it reveals measurable non-NO- non-PGI-mediated endothelium-dependent relaxation otherwise absent in conduit arteries.

Influence of low molecular weight heparin preparations on human internal thoracic artery contraction

Low molecular weight heparins (LMWHs) offer practical and potential pharmacological advantages over unfractionated heparin in multiple applications but have not been studied as vasoactive agents. The purpose of this study was to investigate the effects of two commercial preparations of LMWHs, enoxaparin sodium and nadroparin calcium, on vasoconstriction in the human internal thoracic artery (ITA) in vitro. Samples of redundant ITA segments obtained from 36 patients who underwent coronary artery bypass surgery were cut into 3mm wide rings and suspended in 20 ml organ bath. Activity of ITA rings precontracted with 80 mM KCl, 0.1 microM endothelin-1 (ET-1) and 1 microM norepinephrine (NE) after administration of enoxaparin and nadroparin in accumulative concentration ranging from 0.1 to 13.2 UI AXa/ml were recorded under isometric conditions by means of force transducers with digital output. The contraction after 80 mmol KCl, 0.1 microM ET-1 and 1 microM NE administration was treated as a control. Both studied LMWHs in concentration ranging from 0.12 to 13.2 UI AXa/ml did not change basal tonus and KCl precontracted ITA rings. When used in concentrations higher than 13.2 UI AXa/ml nadroparin but not enoxaparin significantly increased the tension in KCl precontracted arterial rings. In NE and ET-1 precontracted rings enoxaparin and nadroparin caused dose dependent relaxation without significant differences between both preparations. Incubation with nitric oxide blocker-Nomega-NITRO-L-ARGININE (L-NNA) in concentration 0.2 mM caused a significant attenuation of relaxant responses to both studied LMWHs in NE and ET-1 precontracted rings. LMWHs can have vasorelaxant effects on the receptor-mediated ITA vasoconstriction. The results suggest that LMWHs-induced relaxation in the human ITA is at least partially caused by nitric oxide release. Although the vasoactive effects are not the primary advantage of these drugs used as antithrombotics, such effects might have some clinical importance in the treatment and prophylaxis of graft spasm.

The inhibitory action of protamine on human internal thoracic artery contractions: the effect of free hemoglobin.

We investigated the mechanism of the protamine action and the effects of free hemoglobin on protamine-induced responses in endothelium-denuded and-intact human internal thoracic artery (ITA) rings precontracted with phenylephrine (PE) or high KCl. Samples of redundant ITA obtained from patients undergoing a coronary artery bypass graft surgery were cut into 3 mm wide rings and suspended in 20 ml organ baths. Isometric tension was continuously measured with an isometric force transducer connected to a computer-based data acquisition system. Acetylcholine (Ach, 10(-8)-10(-5) M) caused a concentration-dependent relaxation of PE-precontracted ITA rings. Free hemoglobin (0.1 and 0.5 microM) produced a concentration-dependent and significant decrease in sensitivity (pD(2)) and maximal contractility (E(max)) in response to Ach in PE-precontracted ITA rings (P<0.0001). Protamine (50-800 microg/ml), free hemoglobin (0.1 and 0.5 microM), nitric oxide (NO) blocker N(omega)-nitro-L-arginine methyl ester (L-NAME, 100 microM) or soluble guanylate cyclase inhibitor methylene blue (10 microM) administration did not cause a significant alteration on basal tonus of endothelium-intact or -denuded ITA rings. Protamine (50-800 microg/ml) induced concentration-dependent relaxation responses in ITA rings precontracted by either PE or high KCl. There was no difference in sensitivity or maximal response to protamine between the endothelium-intact and -denuded rings. Incubation of endothelium-intact or -denuded ITA rings with L-NAME or free hemoglobin or methylene blue did not cause a significant inhibition on relaxation responses to protamine. ITA ring contractions induced by stepwise addition of calcium to high KCl solution with no calcium were almost completely inhibited by protamine (P<0.0001). It was suggested that protamine induced relaxation responses in human ITA rings is not NO- or endothelium-dependent but seems to depend on the interactions of protamine with calcium influxes and/or calcium release from intracellular stores in this tissue.

Vasoreactivity of arterial grafts in the patient with diabetes mellitus: investigations on internal thoracic artery and radial artery conduits.

Arterial revascularization with either internal thoracic artery (ITA) or radial artery (RA) appears to be particularly attractive in diabetic patients. Previous investigations have shown that endothelial dysfunction and artherosclerosis are seen more often in these patients. The aim of this study was to compare the vasoreactive properties of ITA and RA grafts in diabetic and non-diabetic patients. Arterial rings were harvested from 57 patients who underwent complete arterial revascularization. The patients were divided into a non-diabetic group (I: n = 30) and patients with diabetes mellitus (II: n = 27). Arterial rings of the ITA (I: n = 30; II: n = 27) and RA (I: n = 28; II: n = 19) were mounted on a strain gauge in oxygenated, normothermic Krebs's--Henseleit solution at optimal resting tension. With KCL (80 mM) serving as the control, assessment of force of contraction (norepinephrine), endothelium-dependent relaxation (acetylcholine) and smooth muscle-dependent relaxation (glyceroltrinitrate) were obtained. After KCL, the RA showed a trend to lower maximum contraction forces in diabetics (I: 76 +/- 25 mN; II: 69 +/- 29 mN), which was pronounced in patients with diabetes of more than 10 years duration (55 +/- 23 mN; P = 0.1). Maximum contraction force of the ITA was similar in both groups (I: 41 +/- 20 mN; II: 34 +/- 19 mN) and not influenced by the duration of diabetes. The two groups showed no significant differences of the relative vasoconstriction after norepinephrine in RA (I: 53 +/- 18%; II: 61 +/- 19%) and ITA rings (I: 70 +/- 23%; II: 69 +/- 25%). Also, endothelium-dependent relaxation with acetylcholine in RA (I: 53 +/- 14%; II: 57 +/- 16%) and ITA rings (I: 42 +/- 17%; II: 44 +/- 20%), and smooth muscle relaxation with glyceroltrinitrate of RA (I: 72 +/- 8%; II: 73 +/- 12%) and ITA rings (I: 64 +/- 12%; II: 58 +/- 20%) was comparable in both groups. No influence of duration of the diabetic disease was noted. Although RA rings of patients with a long duration of diabetes have decreased maximum contraction forces, their relative vasoconstriction after norepinephrine, endothelium-dependent relaxation and smooth muscle relaxation was similar to non-diabetic patients. We thus conclude that the RA is an adequate arterial conduit in the patient with diabetes mellitus.

Protamine induces vasorelaxation of human internal thoracic artery by endothelial NO-synthase pathway

Background. Protamine is commonly used in cardiac surgery to reverse the anticoagulant effects of heparin. We investigated the role of different nitric oxide synthase pathways in the response of the human internal thoracic artery to protamine and evaluated whether heparin could prevent this effect. Methods. A tension-recording method was used to obtain baseline measurements of contractions of human internal thoracic artery rings achieved with norepinephrine. Isolated internal thoracic artery rings were suspended in two organ chambers. One contained Krebs-Henseleit solution and served as control. The other contained a heparin or Nω-Nitro-L-arginine (L-NAM, an inhibitor of both endothelial and inducible nitric oxide synthase) or a specific inhibitor of inducible nitric oxide synthase, aminoguanidine. Increasing doses of protamine were added to both chambers and dose-response curves were obtained. Results. Protamine was found to relax contracted internal thoracic arteries 56% ± 4.7% of baseline measurements in a concentration-dependent manner. When L-NAM was added, protamine caused only a slight decrease of tension. There were no differences in the relaxing effect of protamine in the presence of aminoguanidine or heparin. Conclusions. Protamine induces nitric oxide-dependent relaxation of the internal thoracic artery by activation of endothelial nitric oxide synthase pathway. Heparin could not prevent this relaxing effect of protamine.

Characterisation of constrictor endothelin receptors in the human internal thoracic artery and saphenous vein.

We studied the endothelin receptors mediating contraction in the human saphenous vein (SV) and internal thoracic artery (ITA). In the SV, the ET(A)-receptor antagonist BQ123 (1 microM) did not significantly shift the ET-1 concentration-response curve but did cause a parallel shift in the ITA. In the SV, the ET(A)-receptor agonist sarafotoxin 6b (S6b) produced a monophasic concentration-response curve that was antagonised biphasically by BQ123 (0.1-1 microM). In the ITA, S6b was an ineffective agonist with contractions seen only at 3 x 10(-9) M upward. The ET(B)-receptor agonist sarafotoxin 6c (S6c) caused constrictions in only 74% of SV rings and 42% of ITA rings. In the tissues that did respond, S6c caused a monophasic concentration-response curve with a lower maximal response than ET-1. The ET(B) antagonist BQ788 did not antagonise the responses to ET-1 in either the SV or the ITA but did antagonise the responses to S6c in the SV. The results from this study suggest that mainly ET(A) receptors mediate the contractile responses in the human SV and ITA. There is also evidence for an ET(B)-mediated response, although the contractions were much smaller than those elicited by ET-1. We also conclude that the ET(A) receptors mediating responses in these human vessels are atypical because of the different effects of BQ123 on the two vessels.

Direct vasodilator effect of milrinone, an inotropic drug, on arterial coronary bypass grafts

Milrinone is an inotropic drug with vasodilator activity that has been shown to be useful in increasing cardiac output and decreasing wedge pressure. Despite these advantages, it is unknown whether this drug can be used for the treatment of perioperative spasm of coronary bypass grafts. This study was undertaken to investigate the in vitro vascular effect of milrinone on internal thoracic arteries obtained from patients undergoing coronary artery bypass grafting. The results showed that milrinone produced a potent, concentration-dependent, preventive effect on the norepinephrine-induced contraction of internal thoracic arteries, as well as reversing contraction of internal thoracic arteries by receptor-dependent agents, including the thromboxane A 2 mimetic U46619, the vasoconstrictor peptide endothelin-1, and the α 1-adrenal receptor agonist phenylephrine. The relaxing effect of milrinone was weaker, however, on internal thoracic arteries contracted with 25 mmol/L potassium chloride. Comparison of milrinone with other vasodilators, including papaverine, nitroprusside, and glyceryl trinitrate, showed milrinone to be more potent than papaverine but less potent than nitroprusside and glyceryl trinitrate. The inhibitory effect of milrinone on internal thoracic artery contraction appeared as a reduction in contractile force, not as an increase in the values of concentrations of the agonists causing 50% maximal contraction, which indicates that milrinone exerts its vasodilator effect directly on the smooth muscles, not on the membrane receptors. The results also showed no significant difference in relaxing effect between internal thoracic artery rings with and without endothelium. In conclusion, this study provides experimental evidence that milrinone is a potent, endothelium-independent, direct vasodilator of the human internal thoracic artery and provides the scientific rationale for a future clinical trial with this drug for the perioperative treatment of internal thoracic artery spasm in cardiac surgical patients. (J Thorac Cardiovasc 1997;113:108-13)