Internal Mammary Artery Rings Research Articles

Different expression of large-conductance calcium-activated K+ channels in human internal mammary and radial arteries

In this study, we investigated and compared the electrophysiological and molecular properties of large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels between human internal mammary arteries (IMA) and radial arteries (RA). IMA and RA sections were obtained from 79 patients (including 9 females) undergoing coronary artery bypass graft surgery. We examined the effects of K(+) channel blockers tetraethylammonium (TEA), iberiotoxin (IBTX), and 4-aminopyridine (4-AP) on isolated smooth muscle cells (SMCs) using patch clamping. Both TEA (1 mM) and IBTX (0.1 µM) significantly decreased K(+) currents in IMA SMCs and RA SMCs, while 4-AP (1 mM) only had a weak effect. IBTX had a greater K(+)-blocking effect on IMA SMCs than on RA SMCs. Consistently, TEA and IBTX evoked significant constriction of both intact vascular rings. IBTX had a greater constrictor effect on IMA rings (18.5 ± 6.7%, n= 8) than on RA rings (10.6 ± 3.1%, n= 8), P< 0.05. RT-PCR and western blot analysis demonstrated that gene and protein expression of the α-subunit of BK(Ca) channels from IMA was greater than that from RA. The density of BK(Ca) channels is greater in human IMA than in RA resulting in greater BK(Ca) currents in SMCs from IMA. This may partly explain the finding of less spasm in IMA grafts than in RA grafts. Our results may be of value in determining the best anti-spasm agent to use peri-operatively.

Reduced negative surface charge on arterial endothelium explains accelerated atherosclerosis in type 2 diabetic patients

Reduced endothelial surface charge markedly increases the rate of LDL uptake into blood vessels. Previous work in the streptozotocin diabetic rat reported reduced endothelial surface charge. We compared endothelial surface charge density in internal mammary artery rings from patients with type 2 diabetes (n = 12) and from non diabetic patients undergoing coronary artery bypass grafting, and observed a substantial (52%) reduction in the former. This was associated with higher plasma sialic acid levels suggesting loss of sialic acid residues from the glycocalyx as a possible mechanism.

Inhibition of Vasoconstriction by Amlodipine in Human Internal Mammary Arteries Used as Bypass Grafts

Graft spasm remains challenging in coronary artery bypass grafting (CABG). We studied the inhibitory effect of a dihydropyridine calcium antagonist amlodipine in 70 human internal mammary artery (IMA) segments from 34 patients undergoing CABG in organ baths in two ways: the relaxing effect of amlodipine on vasoconstrictor‐induced precontraction by potassium chloride (KCl), human urotensin II (hU‐II), and U46619 and the depressing effect of amlodipine on the contraction. Amlodipine caused full relaxation in KCl (98.0±2.1%)‐ and hU‐II (98.5±2.4%)‐ but not in U46619‐contracted IMA rings (n=8) with 15.5‐fold higher potency to KCl than to hU‐II (EC50: −8.17±0.28 vs. −6.98±0.01 logM, p&lt;0.001). Pretreatment of IMA with plasma‐concentrations of amlodipine (−7.6 and −6.6 logM, n=6) significantly depressed subsequent contraction to KCl (from 20.8±2.5 to 16.6±2.6 mN and from 20.8±2.5 to 7.6±3.0 mN, p&lt;0.05) and hU‐II (from 14.1±4.2 to 11.4±3.7 mN and from 14.1±4.2 to 3.8±2.0 mN, p&lt;0.05) but not to U46619. Thus, in human IMA, amlodipine has a selective inhibitory effect on the vasoconstriction mediated by vasoconstrictors with high potency on KCl and hU‐II and amlodipine may be effectively used in CABG patients for treatment and prevention of graft spasm.Supported by Tianjin Municipal Sci &amp; Tech Com (09ZCZDSF04200); Ministry of Sci &amp; Tech, China (2009DFB30560); Providence SVMF, OR &amp; Hong Kong GRF 465107, 478909.

Alteration of vascular reactivity in diabetic human mammary artery and the effects of thiazolidinediones

Vascular reactivity was investigated in endothelium-denuded human internal mammary artery (IMA) rings from type 2 diabetic patients. It was also investigated whether insulin sensitizer thiazolidinedione drugs, pioglitazone and rosiglitazone, can directly affect the reactivity of IMA. Using organ bath techniques, cumulative concentration-response curves to phenylephrine (PE), KCl, cromakalim (CRO) and sodium nitroprusside (SNP) were constructed in diabetic and non-diabetic IMA rings. Means of maximal responses (% Emax) and pEC50 values (sensitivity) were compared. Emax values and the sensitivity to PE and KCl were increased while K(ATP)-channel-mediated relaxations were reduced significantly in diabetic rings compared with non-diabetic rings (n = 5-12, P < 0.05). No changes were observed for SNP responses (n = 5, P > 0.05). Incubations with pioglitazone (1 and 10 microM) and rosiglitazone (1 and 20 microM), for 30 min, did not affect K(ATP)-channel-mediated relaxations (n = 5 each, P > 0.05). Pioglitazone partly inhibited pre-contractions of PE and KCl at 10 microM, rosiglita-zone did not. Vascular dysfunction observed in diabetic IMA may be of specific importance since they are widely used as coronary bypass material. Thiazolidinedione drugs may not worsen arterial dilatation through K(ATP) channels in ischaemic or hypoxic insults in diabetic patients who are prone to such conditions. Pioglitazone has vasorelaxant property in the grafts.

RhoA/ROK Pathway Related to the Mechanism of Higher Susceptibility to Spasm in RA Than in IMA

By investigating the expression and function of RhoA/Rho kinase pathway in the radial artery (RA), internal mammary artery (IMA), and great saphenous vein (GSV), this study aimed to elucidate the mechanism for a higher susceptibility of spasm in the RA and provide an effective drug candidate to prevent and treat RA spasm. RA, IMA, and GSV that would otherwise have been discarded were collected from 25 patients who underwent coronary artery bypass grafting. Eleven matched rings of RA, IMA, and GSV were used to evaluate the vasodilatory properties of 10(-7-)10(-4) mol/l of fasudil, a Rho-kinase inhibitor, by using in vitro organ chambers. Another 14 matched RA, IMA, and GSV were used to demonstrate the immunohistochemistry (IHC) of RhoA and mRNA of RhoA and Rho kinase. The maximal vasodilation of RA to fasudil was significantly greater than IMA. RhoA protein IHC staining was different in IMA, RA, and GSV (RA > GSV >IMA). The expression of RhoA and Rho kinase mRNA in the RA was significantly greater than in the IMA. The expression of RhoA/Rho kinase mRNA and protein and function in the RA were significantly stronger than in the IMA, suggesting that RhoA/Rho kinase pathway may be one mechanism by which RA is more susceptible to spasm than IMA. Rho kinase inhibitors can be effective drug candidates to prevent and treat vasospasm.

Mechanical Strain Induces Expression of C-Reactive Protein in Human Blood Vessels

C-reactive protein (CRP) is a powerful independent risk factor for cardiovascular diseases. Elevated mechanical strain on vessels induces the local expression of proinflammatory cytokines. We hypothesized that mechanical strain on vessels may induce local CRP expression. Human saphenous vein and internal mammary artery (IMA) rings were stretched in vitro with a mechanical strength of 1, 3, or 5 g. Reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay results showed that mechanical stretching significantly induced CRP mRNA and protein expression in the saphenous vein and IMA rings in a strength-dependent manner reaching a maximum at a mechanical strength of 3 g, but CRP expression returned at strengths of >5 g. In vessels, mechanical strain-induced CRP expression was blocked by two stretch-activated ion channel (SAC) blockers: GdCl(3) and streptomycin. Mechanical strain also increased activation of nuclear factor kappaB (NF-kappaB), which was detected with a nonradioactive NF-kappaB p50/p65 EZ-TFA transcription factor assay. Mechanical strain-induced NF-kappaB activation was blocked by SAC blockers and the NF-kappaB inhibitor (SN50, H-Ala-Ala-Val-Ala-Leu-Leu-Pro-Ala-Val-Leu-Leu-Ala-Leu-Leu-Ala-Pro-Val-Gln-Arg-Lys-Arg-Gln-Lys-Leu-Met-Pro-OH). SN50 also blocked mechanical strain-induced CRP expression in vessels. In conclusion, mechanical strain induces CRP expression in IMAs and saphenous veins by activating the SAC-induced NF-kappaB pathway.

Inhibition of Vascular Constrictor Responses with Acute Statin Treatment is Independent of the Type of Statin, Species or Vascular Bed

Purpose: Previously we have shown that acute statin pretreatment inhibits constrictor responses in isolated rat mesenteric microvessels. The objective of this study is to extend these observations to human large vessels and an isolated heart preparation. Methods: (1) Isolated Human Arteries: Internal mammary artery (IMA) rings from 6 patients undergoing coronary artery bypass grafting, were mounted in an organ bathandendothelial integrity assessedwithacetylcholine. Noradrenaline (NA) concentration–response curves were determined following 60min incubation with either pravastatin (112 nM), simvastatin (61 nM) or vehicle control. Experimentswere repeated in endothelium-denuded vessels or following nitric oxide synthase inhibitor (LNAME; 300 M) treatment. (2) Isolated Rat Beating Heart Preparation: Using a rat Langendorff model pretreated with either vehicle control, p c l R e p c 2 w p r c e C i b s s s

Role of Endothelin-1 Receptor Antagonists in Vasoconstriction Mediated by Endothelin and Other Vasoconstrictors in Human Internal Mammary Artery

The action of antagonists for endothelin type A (ET(A)) and type B (ET(B)) on the vasoconstriction mediated by various vasoconstrictors in the human bypass grafts have not been well-defined. We studied the role of antagonists for both ET(A) and ET(B) receptors in vasoconstriction mediated by endothelin-1 and other vasoconstrictors in the human internal mammary artery (IMA). Isolated IMA rings (n = 192, taken from 49 patients) were studied in organ bath for the interaction between endothelin-1, angiotensin II, U46619, and potassium chloride and the antagonist for ET(A) (BQ-123) or ET(B) (BQ-788). Significant relaxations were observed by BQ-123 (agonist: endothelin-1, 84.9 +/- 7.9%; angiotensin II, 45.5 +/- 5.1%; and U46619, 30.7 +/- 5.7%) or BQ-788 (agonist: endothelin-1, 66.5 +/- 11.3%; angiotensin II, 38.9 +/- 4.2%; and U46619, 30.8 +/- 4.0%), but not to potassium chloride-induced precontraction. Incubation of IMA with BQ-123 or BQ-123 + BQ-788 significantly shifted the concentration-contraction curve to endothelin-1 rightward (p < 0.05 vs control) with effective concentration causing 50% of maximal response (EC50) (-7.59 +/- 0.04 or -7.81 +/- 0.05 vs -8.47 +/- 0.05 log M in the control, p < 0.001), whereas BQ-788 alone did not affect the contraction curve (p = 1.0 vs control). In contrast, none of the endothelin-1 inhibitors and the combination demonstrated significant depression effects on angiotensin II, U46619, or potassium chloride-induced contraction. The present study demonstrates the role of ET(A) and ET(B) antagonists in the endothelin-1-mediated contraction in the human IMA and indicates the dominant role of ET(A) receptors. Although these effects are specific to endothelin-1, cross-action between endothelin-1 and angiotensin II exists. These findings provide useful knowledge for the future development of the clinical antispastic protocol in coronary bypass surgery.

Abstract 1574: Inhibition Of Extracellular Ca 2+ Influx And Rho Kinase Mediate Hypoxic Dilation Of Human Mammary Artery

Objective : Systemic artery dilates in response to decrease in Po 2 , to maintain blood supply to stressed organ. However, the mechanism of hypoxic dilation is still unknown and the effect of hypoxia on human systemic arteries is infrequently studied. Since the internal mammary artery (IMA), a routinely used bypass conduit, is often exposed to low Po 2 that evokes vasospasm and leads to hypo-perfusion of myocardium in peri- and post-operative conditions, we initiated this study to elucidate the mechanism of hypoxic dilation in IMA. Methods &amp; Results : The distal IMA was collected during a CABG surgery in a blinded-fashion and 2–3 mm rings were used in the isometric tone study. The IMA rings pre-contracted with either KCl (30 mM; n=7), a depolarizing agent, or phenylephrine (PE: 10 μM; n=7), a α-adrenergic agonist, relaxed to 34 ± 12% and 10±7%, respectively, of the steady-state pre-hypoxic contraction, in a time-dependent manner by decreasing Po 2 from 140 to 30 torr. The initiation of hypoxic dilation was mediated by opening of K + channels, because IMA pre-contracted with PE relaxed (P=0.027) to 74±12% of the steady-state pre-hypoxic contraction @ 3 minutes by hypoxia and tetraethylammonium acetate (TEA; 10 mM), a K + channel blocker, suppressed (P=0.059) relaxation (97.4±1.6% @ 3 minutes). More interestingly pretreatment of IMA with Y-27362 (10 μM), a Rho kinase inhibitor, shifted Ca 2+ sensitivity to the right, since [Ca 2+ ] o required to induce 50% contraction of IMA by KCl (30 mM) was shifted from 0.3±0.1 mM (control) to 1.1±0.1 mM (Y-27362), and decreased (P=0.043) hypoxic dilation (Y-27362+KCl: 74±14%; n=5 versus KCl: 34±12%; n=7; @ 3 minutes). Hypoxia also shifted Ca 2+ sensitivity and suppressed (P&lt;0.005) steady-state contraction (Normoxia: 4.3±0.1g versus Hypoxia: 1.2±0.6g) evoked by 1.5 mM [Ca 2+ ] o . Conclusion : Our results, therefore, demonstrate that inhibition of extracellular Ca 2+ influx and Rho kinase plays a major role in hypoxic dilation of human artery and we propose that response of IMA conduits to hypoxia could be compromised in hypertension and diabetes in which the Ca 2+ handling and Rho kinase is modulated, leading to hypo-perfusion of myocardium in hypertensive/diabetic patients undergoing CABG surgery. (Supported by AHA Grant #0435070N)

The Gender Differences in the Relaxation to Levosimendan of Human Internal Mammary Artery

The mechanism of the vasorelaxation to levosimendan varies depending on the vascular bed and species studied. Here, we examined the vasorelaxation to levosimendan as well as its modification by various potassium channel antagonists in human internal mammary artery (IMA) obtained from male and female patients. IMA grafts were supplied from 27 male and 19 age-matched female patients undergoing coronary bypass operation. The contraction to noradrenaline and relaxation to levosimendan were studied in IMA rings obtained from both gender. The relaxations to levosimendan were also assessed in the presence of glibenclamide (10 microM), an adenosine triphosphate-sensitive potassium channel (K(ATP)) blocker, or charybdotoxin (100 nM), a calcium-activated potassium channel (K(Ca)) blocker, or 4-aminopyridine(1 mM), a voltage-sensitive potassium channel (K(v)) inhibitor. Concentration-response curves to noradrenaline were not different in IMA rings from either gender. Pretreatment with levosimendan (3 x 10(-7) M) slightly modified the contractions to noradrenaline in both gender. Levosimendan (10(-9)-10(-5) M) produced concentration-dependent relaxation in IMA rings, contracted by noradrenaline (5 x 10(-6) M), from males and females. The vasodilatory effects of levosimendan were more pronounced in the arteries from males (83%) than females (69%), in term of the maximal relaxation (E (max)). Charybdotoxin and glibenclamide significantly inhibited the relaxation to levosimendan in the arteries from males but not in those of females. The vasodilating efficacy of levosimendan and its relaxation mechanism differs between the arteries from males and females, which may have clinical consequences in the treatment of heart failure.

Effect and mechanism of cilnidipine in human internal mammary artery (IMA)

We investigated the efficacy of cilnidipine (CIL), a novel dihydropyridine calcium channel antagonist that possesses stimulating properties on nitric oxide (NO)-cGMP pathway, in human IMA rings (n=66) with comparison to nifedipine (NIF) in organ chambers. Cumulative concentration (−10~−5.5 logM)-relaxation curves (n=6) for CIL with/without ODQ (cGMP inhibitor, 3 μM) and L-NNA (300 μM) and for NIF were established in rings precontracted with K+ (40 mM) or norepinephrine (NE, 10 μM). Concentration-contraction curves for K+ (10~100mM) were also reconstructed after incubation with plasma concentrations of CIL (−6.7 and −7.7 logM) or solvent for 30min. CIL and NIF caused nearly full relaxation (94.7±2.6% vs 100±0%, p>0.05) with similar EC50s (−8.90±0.90 vs −8.46±1.08 logM, p>0.05) on K+-precontracted IMA and limited relaxation (27.3±7.8% vs 39.8±4.6%; EC50: −7.15±0.44 vs −7.14±0.43 log M, p>0.05) against NE. Pretreatment with CIL (−6.7 logM) depressed the K+-induced contraction (59.7±6.7%, p<0.05) and right-shifted the curve (EC50: 95.0±21.5 vs 36.4±12.3 mM, p<0.01). ODQ significantly (39.2±4.7%, p<0.05) and L-NNA (45.8±7.8%, p>0.05) or endothelium-denudation (48.7±8.2%, p>0.05) slightly reduced the relaxation. Thus, CIL significantly inhibits contraction at plasma concentrations and induces relaxation through both endothelium-independent and -dependent mechanisms in the human IMA. (Supported by St. Vincent Medical Foundation, Portland, OR, USA & CUHK 2041164)

Both T-786C and G894T polymorphism of endothelial nitric oxide synthase affect in-vitro endothelium-dependent relaxation of internal mammary artery rings from patients with coronary artery disease

Polymorphisms of endothelial nitric oxide synthase (eNOS) gene in the promoter (T-786C) and exon 7 (G894T) have been suggested to attenuate endothelial function. As it is unknown whether these polymorphisms, on top of classical risk factors, further deteriorate endothelium-dependent vasomotion, we aimed to elucidate the impact of both polymorphisms on the ex-vivo vasomotor function of left internal mammary artery rings from patients with coronary artery disease (CAD) undergoing coronary bypass surgery (CABG). Mammary artery rings from 51 consecutive patients with CAD were obtained during elective CABG. Endothelium-dependent ring relaxation was measured in vitro in an organ chamber using acetylcholine (10 to 3 x 10 mol/l). Polymorphisms were determined by polymerase chain reaction restriction length polymorphism. Thirty-three per cent of patients were positive for the T-786C polymorphism, 25% for the G894T polymorphism, and 18% carried mutated alleles in both loci. Maximal acetylcholine-induced ring relaxation was 46.7+/-3.2% in T-786C, 59.6+/-4.2% in G894T, and 66.7+/-7.4% in T-786C/G894T compared with 94.9+/-2.0% in wild-type subjects (P<0.05 versus T-786C, G894T, T-786C/G894T). Patients positive for an eNOS polymorphism with more than three cardiovascular risk factors displayed a further attenuation of acetylcholine-mediated relaxation (45+/-6 %) compared with having up to three risk factors (59+/-3%, P<0.05). In patients with CAD, in-vitro assessed endothelium-dependent relaxation of mammary arteries was significantly impaired in those positive for the T-786C or the G894T eNOS polymorphism. These results suggest that the presence of either one of the eNOS polymorphisms deteriorated endothelium-dependent vasodilatory capacity of large conduit vessels on top of classical risk factors in patients with CAD.

Endothelial Protection, AT 1 Blockade and Cholesterol-Dependent Oxidative Stress

Statins and angiotensin type 1 (AT1) receptor blockers reduce cardiovascular mortality and morbidity. In the Endothelial Protection, AT1 blockade and Cholesterol-Dependent Oxidative Stress (EPAS) trial, impact of independent or combined statin and AT1 receptor blocker therapy on endothelial expression of anti-atherosclerotic and proatherosclerotic genes and endothelial function in arteries of patients with coronary artery disease were tested. Sixty patients with stable coronary artery disease undergoing elective coronary artery bypass grafting (CABG) surgery were randomized 4 weeks before surgery to: (A) control without inhibition of renin-angiotensin system or statin; (B) statin (pravastatin 40 mg/d); (C) AT1 blockade (irbesartan 150 mg/d); or (D) combination of statin and AT1 blocker in same dosages. Primary end point was a priori therapy-dependent regulation of an anti-atherosclerotic endothelial expression quotient Q including mRNA expression (in arbitrary units measured by real-time polymerase chain reaction) of endothelial nitric oxide synthase and C-type natriuretic peptide, divided by expression of oxidized low-density lipoprotein receptor LOX-1 and NAD(P)H oxidase subunit gp91phox in left internal mammary arteries biopsies obtained by CABG surgery; 49 patients completed the study. Statin therapy increased lnQ from 3.2+/-0.4 to 4.4+/-0.4 significantly versus control. AT(1) blockade showed a trend to increase lnQ to 4.2+/-0.5. Combination of statin and AT1 blocker further increased lnQ to 5.1+/-0.6, but a putative interaction of both therapies in lnQ was not significant. Furthermore, preoperative therapy with statin, AT1 blocker and their combination improved endothelial function in internal mammary artery rings. Statin and AT1 blocker therapy independently and in combination improve an anti-atherosclerotic endothelial expression quotient and endothelial function.

THERMAL PRECONDITIONING PROTECTS THE HUMAN INTERNAL MAMMARY ARTERY FROM HYPOXIA/RE‐OXYGENATION‐INDUCED DAMAGE

1. Preconditioning has been demonstrated to ameliorate ischaemia/reperfusion injury in several cells and tissues. Therefore, in the present study we investigated whether preconditioning of human bypass grafts, internal mammary artery (IMA) and saphenous vein (SV) induces heat shock protein (Hsp) expression and reduces apoptosis in response to subsequent hypoxia/re-oxygenation damage in both vessels. 2. Internal mammary artery and SV rings, obtained from 30 patients (median age 66.5 years) undergoing coronary artery bypass grafting, were either incubated for 30 min at 42 degrees C (preconditioned) or kept in a standard incubator at 37 degrees C (not preconditioned). Six hours later, graft segments were exposed to 90 min hypoxia followed by a 30 min re-oxygenation period. Western blot, real-time quantative polymerase chain reaction analysis and apoptosis detection by the Terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labelling method were performed. 3. Heat-preconditioned IMA showed significantly increased protein expression of Hsp72 after hypoxia/re-oxygenation treatment compared with controls (median 9.1 vs 5.0 microg/mg total protein; P = 0.048). Expression of Hsp73 was weak and Hsp60 was not detectable in the IMA. 4. In the SV, neither protein nor mRNA expression of Hsp were significantly different between preconditioned and not preconditioned veins. 5. There were significantly fewer apoptotic cells in the intima of the preconditioned compared with not preconditioned IMA (P = 0.041) after hypoxia/re-oxygenation injury, whereas in the SV apoptosis was not significantly prevented by preconditioning. 6. Mild heat preconditioning before hypoxia/re-oxygenation injury is a stimulus for Hsp72 protein expression and a reduction in apoptosis in the human IMA.

Leptin is an endothelial-independent vasodilator in humans with coronary artery disease: evidence for tissue specificity of leptin resistance

We sought to define the mechanisms and correlates of leptin's vascular actions in humans with coronary artery disease. In 131 patients (age 65.7+/-0.7 years mean+/-SEM), ex vivo vascular reactivity to leptin (10(-13)-10(-7) M) was assessed in saphenous vein (SV) rings. Leptin led to SV relaxation (maximal relaxation 24.5+/-1.6%). In separate experiments, relaxation to leptin was unaffected by L-NMMA (17.4+/-3.4 vs.17.8+/-3.3%, P = 0.9) or endothelial denudation (17.4+/-4.4 vs. 22.5+/-3.0%, P = 0.4). We explored the possibility that leptin's vascular effects are mediated via smooth muscle hyperpolarization. In the presence of KCl (30 mmol/L) to inhibit hyperpolarization, the vasodilator effect of leptin was completely blocked (0.08+/-4.1%, P < 0.001 vs. control). Similar results were demonstrated in internal mammary artery rings. The only independent correlate of leptin-mediated vasodilatation was plasma TNF-alpha (r = 0.25, P < 0.05). Neither body mass index nor waist circumference correlated with leptin-mediated vasorelaxation. This lack of a correlation with markers of total body fat/fat distribution suggests that leptin resistance may not extend to the vasculature. Leptin is a vasoactive peptide in human SV and internal mammary artery. Its action is not nitric oxide or endothelial-dependent. Markers of body fat did not correlate with leptin-mediated vasodilatation, raising the intriguing possibility of selective resistance to leptin's actions.

Effect of Resveratrol on Nitrate Tolerance in Isolated Human Internal Mammary Artery

The present study aims to examine whether resveratrol, a natural antioxidant present in red wine, restores the tolerance to nitroglycerin (GTN) on isolated human internal mammary artery (IMA), using an in vitro model of nitrate tolerance. IMA rings were obtained from 53 male patients undergoing coronary bypass operation. Nitrate tolerance was induced by incubating the artery ring with 100 microM GTN for 90 minutes. Concentration-response curves to GTN (10(-9) to 10(-4) M) were obtained on IMA rings precontracted with noradrenaline. A low concentration (5 microM) of lucigenin was used as a tool to measure superoxide production in IMA segments. GTN produced concentration-dependent relaxation in isolated human IMA rings. Preexposure of artery rings to GTN reduced the relaxations to GTN [E(max) values: 105 +/- 2% and 76 +/- 3%, n = 10 to 12, P < 0.05; EC(50) values (-log M): 6.72 +/- 0.05 and 4.95 +/- 0.06, P < 0.05, respectively]. Relaxation to sodium nitroprusside remained unchanged. Diminished relaxation to GTN is partially restored after removing endothelium or L(G)-nitro-L-arginine (L-NOARG, 10 M) or superoxide dismutase (20 and 200 U/mL) or catalase (200 U/mL) pretreatments. Pretreatments with resveratrol (1, 10, and 20 microM) for 20 minutes relatively improved the reduced relaxation to GTN in tolerant IMA rings. Coadministration of L-NOARG with resveratrol did not abolish the beneficial effect of resveratrol on nitrate tolerance. The inhibitory effect of resveratrol on GTN-induced tolerance was not abolished in arterial rings without endothelium. Exposure to GTN increased superoxide production in IMA segments with endothelium. Endothelium denudation, L-NOARG, or superoxide dismutase pretreatments markedly inhibited the increased superoxide production in tolerant arteries. Resveratrol (1 and 10 microM) almost completely abolished basal or NAD(P)H-stimulated superoxide production in tolerant and nontolerant arteries. Vascular tolerance to GTN, in in vitro tolerant human IMA rings, can be induced by endothelial superoxide anions. Resveratrol partially restored the reduced relaxation to GTN by inhibiting NAD(P)-derived superoxide production in endothelium.

Characterization of Beta3-Adrenoceptors in Human Internal Mammary Artery and Putative Involvement in Coronary Artery Bypass Management

The aim of the present study was to analyze whether beta3-adrenoceptors (beta3-ARs) were effectively present and functional in the human internal mammary artery (IMA). The beta1- and beta2-adrenoceptors classically mediate the relaxant effects of catecholamines in the vessels. In vitro and in vivo studies performed in various animal species described vasodilating effects due to activation of a third beta-ARs subtype (beta3). Reverse transcription-polymerase chain reaction analysis, Western blot experiments, and pharmacological studies were carried out in human IMA samples harvested from 27 patients undergoing coronary bypass surgery. The beta3-ARs messenger ribonucleic acid and protein were detected in intact IMA, but were absent in endothelium-free samples. This finding was confirmed by immunohistochemical experiments. In organ baths, a beta3-AR agonist, SR 58611A, induced an endothelium-dependent relaxation of phenylephrine-precontracted IMA rings. This vasodilation was not modified by beta1/beta2-AR antagonists, but was greatly altered in the presence of L-748,337, a selective human beta3-AR antagonist. Moreover, the inhibition of nitric oxide (NO) synthases abolished the beta3-adrenergic vasodilation, suggesting the involvement of a NO-signaling pathway. Those results demonstrated the presence of beta3-ARs in the endothelial layer of human IMA. The present work highlights the role of beta3-ARs in vasomotor control of IMA and opens new fields of investigation in coronary bypass graft management, heart failure, and hypertension.

Comparison of the Vasodilatory Effect of Nadroparin, Enoxaparin, Dalteparin, and Unfractioned Heparin in Human Internal Mammary Artery

The aim of this study was to investigate whether the low-molecular-weight heparins (LMWHs) (eg, nadroparin, enoxaparin, and dalteparin) cause a vasodilatory effect in human internal mammary artery (IMA) and to further compare its effect with unfractioned heparin (UFH). Samples of redundant IMA obtained from 20 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. LMWHs (0.5-6 U/mL) caused a concentration-dependent relaxation in the endothelium-intact human IMA rings, which were precontracted with Phe (10(-6) M) (P < 0.05). The vasodilator potency of LMWHs seems to be nearly similar while the maximal effect produced by LMWHs was less pronounced compared with that produced by UFH. Removal of endothelium totally abolished the responses of human IMA to LMWHs as well as UFH (P < 0.05). LMWHs-induced vasodilator effect was significantly attenuated by Nomega-nitro-L-arginine methyl ester (L-NAME, 10(-4) M) but not indomethacin (10(-5) M). Our results have shown that LMWHs cause a dose-dependent relaxation in human IMA but are less effective than that produced by UFH. The vasorelaxant effects induced by each of LMWH are nearly similar and seem to be via endothelium-dependent mechanisms, including generation of nitric oxide.

Inflammation and Endothelial Function

Circulating concentrations of the sensitive inflammatory marker C-reactive protein (CRP) predict future cardiovascular events, and CRP is elevated during sepsis and inflammation, when vascular reactivity may be modulated. We therefore investigated the direct effect of CRP on vascular reactivity. The effects of isolated, pure human CRP on vasoreactivity and protein expression were studied in vascular rings and cells in vitro, and effects on blood pressure were studied in rats in vivo. The temporal relationship between changes in CRP concentration and brachial flow-mediated dilation was also studied in humans after vaccination with Salmonella typhi capsular polysaccharide, a model of inflammatory endothelial dysfunction. In contrast to some previous reports, highly purified and well-characterized human CRP specifically induced hyporeactivity to phenylephrine in rings of human internal mammary artery and rat aorta that was mediated through physiological antagonism by nitric oxide (NO). CRP did not alter endothelial NO synthase protein expression but increased protein expression of GTP cyclohydrolase-1, the rate-limiting enzyme in the synthesis of tetrahydrobiopterin, the NO synthase cofactor. In the vaccine model of inflammatory endothelial dysfunction in humans, increased CRP concentration coincided with the resolution rather than the development of endothelial dysfunction, consistent with the vitro findings; however, administration of human CRP to rats had no effect on blood pressure. Pure human CRP has specific, direct effects on vascular function in vitro via increased NO production; however, further clarification of the effect, if any, of CRP on vascular reactivity in humans in vivo will require clinical studies using specific inhibitors of CRP.

Ex-vivo effect of roxithromycin on human and rat arterial vasoactivity

Prior studies have suggested that inflammation and possibly bacterial infections play a role in atherogenesis and in the clinical pathogenesis of cardiovascular diseases. Treatment with the macrolide antibiotics has been associated with improved outcome from cardiovascular disease, although the mechanism through which they exert their effects may be unrelated to their antibiotic properties. Drugs that exert a vasodilator effect on arteries have been associated with attenuated atherogenesis and improved outcome from cardiovascular disease. To determine the effect of the macrolide, roxithromycin (RX), on arterial vasoactivity. Human internal mammary artery (IMA) and rat thoracic aorta (TA) rings were placed in organ chambers and contracted with norepinephrine (NE). Endothelial and smooth muscle integrity were assessed using acetylcholine (ACh) and nitroprusside (SNP), respectively. After restabilization, the rings were exposed to 10(-6) M NE, followed by increasing concentrations of RX (10(-7)-10(-4) M), then by 10(-5) M SNP. The mechanism of RX action was tested in rats using solutions containing 10(-6) M L-NAME, a nitric oxide synthase inhibitor; 10(-6) M calcium ionophore (Ca), a calcium channel agonist; 10(-6) M indomethacin (Indo), a prostaglandin inhibitor; 10(-6) M glibenclamide (Glib), a membrenal ATP-sensitive-K(+)-channel inhibitor; 10(-6) M 5-hydroxydecanoic acid (5-HD), a mitochondrial ATP-sensitive-K(+)-channel inhibitor. Human IMAs and rat TAs exhibited similar contraction in response to NE and relaxation in response to RX, and ACh. RX-related relaxation was dose dependent (4.5+/-1 to 15+/-3%), similar to ACh, and lower than SNP. Relaxation was significantly reduced in the presence of Ca, L-NAME, and 5-HD (P<0.005). Glib and Indo had no effect on relaxation. Lower levels of relaxation in response to RX were observed in TAs without endothelium (P<0.005). RX exerts a mild endothelium-dependent vasodilatory effect mediated by calcium, mitochondrial K(+)-ATP channels and NO production, possibly explaining part of its mild salutary clinical effects.